CTP : phosphocholine cytidylyltransferase inhibition by ceramide via PKC-alpha, p38 MAPK, cPLA(2), and 5-lipoxygenase

In a companion paper (Vivekananda J, Smith D, and King RT. Am J Physiol Lun g Cell Mol Physiol 281: L98-L107, 2001), we demonstrated that tumor necrosi s factor (TNF)-alpha inhibited the activity of CTP:phosphocholine cytidylyl transferase (CT), the rate-limiting enzyme in the de novo synthesis of phos phatidylcholine (PC), and that its actions were likely exerted through a me tabolite of sphingomyelin. In this paper, we explore the signaling pathway employed by TNF-alpha using C-2 ceramide as a cell-penetrating sphingolipid representative of the metabolites induced by TNF-alpha. We found that in H 441 cells, as reported in other cell types, cytosolic phospholipase A(2) (c PLA(2)) is activated by TNF-alpha. We also observed that the inhibiting act ion of C-2 ceramide on CT requires protein kinase C-alpha, p38 mitogen-acti vated protein kinase, and cPLA2. The actions of Ca ceramide on CT activity can be duplicated by adding 2 muM lysoPC to these cells. Furthermore, we fo und that the effects of C-2 ceramide are dependent on 5-lipoxygenase but th at cyclooxygenase II is unimportant. We hypothesize that CT activity is inh ibited by the lysoPC generated as a consequence of the activation of cPLA(2 ) by protein kinase C-alpha and p38 mitogen-activated protein kinase. The o ther product of the activation of cPLA(2), arachidonic acid, is a substrate for the synthesis of leukotrienes, which raise intracellular Ca2+ levels a nd complete the activation of cPLA(2).