Potential roles of extracellular signal-regulated kinase but not p38 during myeloid differentiation of U937 cells stimulated by cytokines: augmentation of differentiation via prolonged activation of extracellular signal-regulated kinase.
OBJECTIVE
To clarify the signaling mechanism of human myeloid differentiation by hematopoietic growth factors and cytokines, we investigated the role of extracellular signal-regulated kinase (ERK) during the differentiation of human monoblastic U937 cells stimulated by granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor (TNF).


METHODS
Myeloid differentiation was evaluated by morphology, function (respiratory burst activity), and cell surface expression of adhesion molecule (CD11b), and activation of ERK and/or p38 was determined by Western blotting and/or in vitro kinase assay. Inhibition of the ERK pathway was performed using PD98059, a specific inhibitor of this pathway.


RESULTS
U937 cells were induced to be differentiated by the combination of GM-CSF and TNF, but only minimally by either cytokine alone. Transient phosphorylation and activation of ERK was induced by both GM-CSF alone and combination of the two cytokines, whereas sustained phosphorylation and activation was induced only by the combination. In addition, PD98059, a specific inhibitor of ERK pathway, almost completely abolished this prolonged phosphorylation of ERK and completely blocked differentiation. In contrast, both TNF alone and cytokine combination equivalently phosphorylated p38 in U937 cells, which was dissociated from differentiation, and a specific inhibitor of p38 (SB203580) did not inhibit differentiation.


CONCLUSIONS
The results indicate potential roles of sustained activation of ERK but not of p38 in the signaling pathways for human myeloid differentiation in U937 cells synergistically stimulated by the two physiologic cytokines GM-CSF and TNF.
Okuma
Emiko
E
Department of Hematology, Research Institute, International Medical Center of Japan, Tokyo, Japan.
Inazawa
Yuko
Y
Saeki
Kumiko
K
Yuo
Akira
A
D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D016207:Cytokines / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology*; D006801:Humans; D020935:MAP Kinase Signaling System / Q000502:physiology; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D020298:U937 Cells; D048051:p38 Mitogen-Activated Protein Kinases
D016207:Cytokines; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases