Ceramide-1-phosphate activates cytosolic phospholipase A2alpha directly and by PKC pathway.
Ceramide-1-phosphate (C1P), a novel bioactive sphingolipid, is implicated in the vital cellular processes such as cell proliferation and inflammation. The role of C1P on activity of cytosolic phospholipase A2alpha (cPLA2alpha), a key enzyme for the release of arachidonic acid (AA) and prostanoids, has not been well elucidated. In this study, we investigated the effect of C1P on the release of AA from L929 cells and a variant, which lacks cPLA2alpha expression, C12 cells. C1P at 30 microM alone induced AA release from L929 cells without an increase in intracellular Ca2+ concentration. C1P-induced AA release was marginal in C12 cells, and treatment with an intracellular Ca2+ chelator (BAPTA-AM) or an inhibitor of cPLA2alpha (2 microM pyrrophenone) decreased C1P-induced AA release in L929 cells. C1P increased the enzymatic activity of cPLA2alpha over two-fold in the presence of Ca2+. C1P triggered the translocation of cPLA2alpha and its C2 domain from the cytosol to the perinuclear region in CHO-K1 cells. Interestingly, C1P at 10 microM synergistically enhanced ionomycin-induced AA release from L929 cells. The AA release induced by C1P with and without ionomycin decreased by treatment with protein kinase C (PKC) inhibitor (10 microM GF109203X) and in the PKC-depleted cells. C1P at 10 microM stimulated the translocation of PKC (alpha and delta) from the soluble to the membrane fractions. We propose that C1P stimulates AA release via two mechanisms; direct activation of cPLA2alpha, and the PKC-dependent pathway.
Nakamura
Hiroyuki
H
Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675, Japan.
Hirabayashi
Tetsuya
T
Shimizu
Masaya
M
Murayama
Toshihiko
T
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D002460:Cell Line; D002518:Ceramides / Q000494:pharmacology*; D003600:Cytosol / Q000187:drug effects* / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D004338:Drug Combinations; D004789:Enzyme Activation; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D054513:Group IV Phospholipases A2; D051379:Mice; D010741:Phospholipases A / Q000096:biosynthesis*; D014176:Protein Biosynthesis / Q000187:drug effects; D011493:Protein Kinase C / Q000096:biosynthesis*
D002518:Ceramides; D004338:Drug Combinations; C065576:ceramide 1-phosphate; D016718:Arachidonic Acid; D011493:Protein Kinase C; D010741:Phospholipases A; D054513:Group IV Phospholipases A2