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.
Footnotes
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This work was supported by National Institutes of Health Grant 19134-26 (to K.U.M.), an American Heart Association's Beginners grant-in-aid (to M.M.M.), and a Health Sciences fellowship (to N.P.).
- Abbreviations:
- MAP
- mitogen-activated protein
- ERK
- extracellular regulated kinase
- cPLA2
- cytosolic phospholipase A2
- CaM kinase II
- Ca2+/calmodulin-dependent kinase II
- FBS
- fetal bovine serum
- ATK
- arachidonyl trifluoromethyl ketone
- MAFP
- methyl arachidonyl fluorophosphonate
- MTT
- 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
- PBS
- phosphate-buffered saline
- BSA
- bovine serum albumin
- PAGE
- polyacrylamide gel electrophoresis
- DMEM
- Dulbecco's modified Eagle's medium
- LPC
- lysophosphatidyl choline
- Received December 26, 2000.
- Accepted March 27, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
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