Effects of ceramide, ceramidase inhibition and expression of ceramide kinase on cytosolic phospholipase A2alpha; additional role of ceramide-1-phosphate in phosphorylation and Ca2+ signaling.
Ceramide and the metabolites including ceramide-1-phosphate (C1P) and sphingosine are reported to regulate the release of arachidonic acid (AA) and/or phospholipase A(2) (PLA(2)) activity in many cell types including lymphocytes. Recent studies established that C1P, a product of ceramide kinase, interacts directly with Ca(2+) binding regions in the C2 domain of alpha type cytosolic PLA(2) (cPLA(2)alpha), leading to translocation of the enzyme from the cytosol to the perinuclear region in cells. However, a precise mechanism for C1P-induced activation of cPLA(2)alpha has not been well elucidated; such as the phosphorylation signal caused by the extracellular signal-regulated kinases (ERK1/2) pathway, a downstream of the protein kinase C activation with 4beta-phorbol myristate acetate (PMA), is required or not. In the present study, we showed that the increase in intracellular ceramide levels (exogenously added cell permeable ceramides and an inhibition of ceramidase by (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol and the increase in C1P formation by transfection with the vector for human ceramide kinase significantly enhanced the Ca(2+) ionophore (A23187) -induced release of AA via cPLA(2)alpha's activation in CHO cells. Ceramides did not show additional effects on the release from the cells treated with the inhibitor of ceramidase. Ceramides and C2-C1P neither had effect on the intracellular mobilization of Ca(2+) nor the phosphorylation of cPLA(2)alpha in cells. A23187/PMA-induced release of AA was enhanced by ceramides and C2-C1P and by expression of ceramide kinase. Our findings suggest that C1P is a stimulatory factor on cPLA(2)alpha that is independent of the Ca(2+) signal and the PKC-ERK-mediated phosphorylation signal.
Shimizu
Masaya
M
Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba 260-8675, Japan.
Tada
Eiko
E
Makiyama
Tomohiko
T
Yasufuku
Kana
K
Moriyama
Yuta
Y
Fujino
Hiromichi
H
Nakamura
Hiroyuki
H
Murayama
Toshihiko
T
D021581:Active Transport, Cell Nucleus / Q000187:drug effects / Q000502:physiology; D000818:Animals; D016466:CHO Cells; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D055572:Ceramidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002518:Ceramides / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D006224:Cricetinae; D003412:Cricetulus; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000187:drug effects / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D054513:Group IV Phospholipases A2 / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D010766:Phosphorylation / Q000187:drug effects; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D011493:Protein Kinase C / Q000187:drug effects / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D015854:Up-Regulation / Q000235:genetics
D002518:Ceramides; C065576:ceramide 1-phosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C066324:ceramide kinase; D011493:Protein Kinase C; D048049:Extracellular Signal-Regulated MAP Kinases; D054513:Group IV Phospholipases A2; D055572:Ceramidases