Abstract

The signaling mechanisms downstream of growth factor-stimulated proliferation in myeloid leukemia cells have not yet been fully elucidated. Recent evidence suggests that alternate pathways to the mitogen-activated protein kinase cascade are required. We have previously shown that Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II) activates cytosolic phospholipase A₂ (cPLA₂), which is involved in the proliferation of vascular smooth muscle cells. In the present study, the contribution of this pathway was investigated in the proliferation of U-937 myeloid leukemia cells. In U-937 cells, fetal bovine serum (FBS)-induced proliferation was attenuated by CaM kinase II inhibitor KN-93 but not by its inactive analog KN-92. Inhibitors of cPLA₂ (methyl arachidonyl fluorophosphonate and arachidonyl trifluoromethyl ketone) also reduced proliferation of U-937 cells. FBS-induced proliferation was also attenuated by cotransfection with cPLA₂ antisense oligonucleotides. These results suggest a role for CaM kinase II and cPLA₂ in the proliferation of U-937 cells. FBS stimulated CaM kinase II and cPLA₂activities in a time-dependent manner. Moreover, FBS-stimulated phosphorylation and activation of cPLA₂ activation was inhibited by KN-93. FBS-stimulated phosphorylation of CaM kinase II was blocked by KN-93 but not by cPLA₂ inhibitors, suggesting that CaM kinase II activates cPLA₂. The products of phospholipid hydrolysis produced by cPLA₂, lysophosphatidylcholine but not arachidonic acid, increased [³H]thymidine incorporation in U-937 cells. These data suggest that exposure of U-937 cells to FBS promotes phosphorylation and activation of CaM kinase II, leading to stimulation of cPLA₂ and generation of lysophosphatidylcholine and resultant proliferation of these cells.