Expression of cytosolic phospholipase A2 alpha in murine C12 cells, a variant of L929 cells, induces arachidonic acid release in response to phorbol myristate acetate and Ca2+ ionophores, but not to tumor necrosis factor-alpha.

Tumor necrosis factor-alpha (TNFalpha)-induced cell death is regulated through the release of arachidonic acid (AA) by group IVA cytosolic phospholipase A2 (cPLA2alpha) in the murine fibroblast cell line L929. However, the signaling pathway by which TNFalpha activates cPLA2alpha remained to be solved. We examined AA release in L929 cells, in a variant of L929 (C12 cells) lacking cPLA2alpha, and in C12 cells transfected with cPLA2alpha expression vectors. In transient and stable clones of C12 cells expressing cPLA2alpha, Ca2+ ionophore A23187 and phorbol myristate acetate (PMA) stimulated AA release within 90 min, although no response to TNFalpha was observed within 6 h. These results suggest that C12 cells may lack the components necessary for TNFalpha-induced AA release, in addition to cPLA2alpha. PMA is known to stimulate AA release via phosphorylation of Ser505 in cPLA2alpha by activating extracellular signal-regulated kinases (ERK1/2). However, PMA-induced AA release from C12 cells expressing mutant cPLA2alpha S505A (mutation of Ser505 to Ala), which is not phosphorylated by ERK1/2, was similar to that from L929 cells and C12 cells expressing wild-type cPLA2alpha. The role of Ser505 phosphorylation in AA release induced by PMA is also discussed.