Role of protein kinase C and the Na+/H+ antiporter in suppression of apoptosis by granulocyte macrophage colony-stimulating factor and interleukin-3.
Granulocyte macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3) suppress apoptosis in hemopoietic cells, a process of active cell death characterized by the degradation of genomic DNA into oligonucleosomic fragments. The present study was therefore initiated with the view that the two growth factors may trigger the same early events in the cell, leading to suppression of apoptosis. We provide evidence here for a role of protein kinase C and of the Na+/H+ antiporter in the signal transduction pathways activated by binding of GM-CSF or IL-3 to their respective receptors, resulting in suppression of apoptosis in target cells. First, kinetic studies indicate that the process is irreversible after two hours of deprivation. The suppression of apoptosis by GM-CSF and IL-3 is dose-dependent, with half-efficient concentrations that are in the range of the dissociation constants of the high affinity GM-CSF or IL-3 receptor, respectively. Second, the use of three inhibitors of protein kinase C (PKC), H7, staurosporine, and sphingosine, in concentrations that are below their toxicity limits, revert the suppression of apoptosis by IL-3 and GM-CSF. Conversely, the use of 12-O-tetradecanoylphorbol-13-acetate (TPA), a PKC activator, allows a bypass of receptor activation in suppression of apoptosis. Western blotting of cytosolic and membrane proteins indicate that exposure of the cells to GM-CSF, IL-3, or TPA results in translocation of PKC to the cell membrane. Our data, therefore, indicate that the activation of PKC is important in suppression of apoptosis by GM-CSF and IL-3. Third, the two amiloride derivatives 5-(N,N-hexamethylene) and 5-(N-ethyl-N-isopropyl)amiloride that specifically block the function of the Na+/H+ antiport also revert the protective effect of GM-CSF, IL-3, and TPA on MO7-E cells. Further, exposure of the cells to GM-CSF, IL-3, or TPA results in sustained pHi alkalinizatio, which is abrogated when the cells are preincubated with 5-(N-ethyl-N-isopropyl)amiloride, a specific inhibitor of the antiport. Preincubation of the cells with staurosporine, a PKC inhibitor, also significantly reduces the effect of GM-CSF or IL-3 on pHi. Taken together, our data indicate that a functional antiport is required in suppression of apoptosis by GM-CSF, IL-3, or TPA. Furthermore, our results are consistent with the view that GM-CSF or IL-3 receptor activation initiates the sequential activation of PKC and of the Na+/H+ antiporter, resulting in suppression of apoptosis in target cells.
Rajotte
D
D
Department of Medicine, University of Montreal, Quebec, Canada.
Haddad
P
P
Haman
A
A
Cragoe
E J
EJ
Hoang
T
T
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000470:Alkaloids / Q000494:pharmacology; D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism*; D016923:Cell Death / Q000187:drug effects*; D002999:Clone Cells; D004273:DNA, Neoplasm / Q000378:metabolism; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D007377:Interleukin-3 / Q000378:metabolism / Q000494:pharmacology*; D007546:Isoquinolines / Q000494:pharmacology; D007700:Kinetics; D007947:Leukemia, Megakaryoblastic, Acute; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism; D016185:Receptors, Interleukin-3 / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D017923:Sodium-Hydrogen Exchangers; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000470:Alkaloids; D002352:Carrier Proteins; D004273:DNA, Neoplasm; D007377:Interleukin-3; D007546:Isoquinolines; D010879:Piperazines; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D016185:Receptors, Interleukin-3; D011994:Recombinant Proteins; D017923:Sodium-Hydrogen Exchangers; D000584:Amiloride; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D011493:Protein Kinase C; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate