Granulocyte colony-stimulating factor-induced terminal maturation of human myeloid cells is specifically associated with up-regulation of receptor-mediated function and CD10 expression.

The acute promyelocytic leukemia cell line NB4 was differentiated by all-trans retinoic acid (ATRA), which enhanced the superoxide-producing capacity stimulated by the chemotactic peptide and phorbol ester in this cell line. Granulocyte colony-stimulating factor (G-CSF) by itself had no effect on NB4 cells but exerted additional enhancing effects on the respiratory burst activity in the presence of ATRA. This finding was not due to the induction of G-CSF receptor by ATRA, because NB4 cells expressed abundant G-CSF receptor with or without ATRA. Unlike ATRA, G-CSF enhanced superoxide release stimulated by the chemotactic peptide but not by phorbol ester. In addition, G-CSF but not ATRA attenuated cell death and enhanced survival during differentiation. Cell surface expression of the chemotactic peptide receptors CD33 and CD10 but not of CD11b and CD11c was up-regulated by ATRA plus G-CSF far more profoundly than by ATRA alone. Fundamentally identical but slightly different phenomena for the cell surface expression of CD33 and CD10 were observed in the normal human bone marrow mononuclear cells; G-CSF induced CD10 even in the absence of ATRA and down-regulated CD33 in normal cells. The present results indicate that G-CSF-induced terminal maturation of human myeloid cells is associated with up-regulation of receptor-mediated function and CD10 expression.

Lipid droplet formation in human myeloid NB4 cells stimulated by all trans retinoic acid and granulocyte colony-stimulating factor: possible involvement of peroxisome proliferator-activated receptor gamma.

All trans retinoic acid (ATRA), a differentiation inducer for human myeloid NB4 cells, induced accumulation of lipid droplet as determined by positivity of Nile Red and Oil Red O in this cell line. Granulocyte colony-stimulating factor (G-CSF), although not having detectable effect by itself, exerted the additive effects on lipid droplet formation in NB4 cells when combined with ATRA. mRNA analysis for peroxisome proliferator-activated receptors (PPARs) revealed the initial transient downregulation followed by upregulation of the transcript for PPARgamma2, a master molecule for adipogenesis, and upregulation of PPARalpha. BADGE, a synthetic antagonist for PPARgamma, potently inhibited lipid droplet formation in NB4 cells stimulated by ATRA and/or G-CSF, but not the functional differentiation of the cells by ATRA and/or G-CSF. These results suggest that ATRA and G-CSF induce lipid droplet formation via certain PPARgamma-mediated specific mechanisms in human myeloid NB4 cells during functional differentiation.

Oxidation-triggered c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein (MAP) kinase pathways for apoptosis in human leukaemic cells stimulated by epigallocatechin-3-gallate (EGCG): a distinct pathway from those of chemically induced and receptor-mediated apoptosis.

We investigated intracellular signalling pathways for apoptosis induced by epigallocatechin-3-gallate (EGCG) as compared with those induced by a toxic chemical substance (etoposide, VP16) or the death receptor ligand [tumour necrosis factor (TNF)]. EGCG as well as VP16 and TNF induced activation of two apoptosis-regulating mitogen-activated protein (MAP) kinases, namely c-Jun N-terminal kinase (JNK) and p38 MAP kinase, in both human leukaemic U937 and OCI-AML1a cells. In U937 cells, the apoptosis and activation of caspases-3 and -9 induced by EGCG but not VP16 and TNF were inhibited with SB203580, a specific inhibitor of p38, while those induced by EGCG and VP16 but not TNF were inhibited with SB202190, a rather broad inhibitor of JNK and p38. In contrast, the EGCG-induced apoptosis in OCI-AML1a cells was resistant to SB203580 but not to SB202190. Unlike TNF, EGCG did not induce the activation of nuclear factor-kappaB but rather induced the primary activation of caspase-9. N -Acetyl-L-cysteine (NAC) almost completely abolished apoptosis induced by EGCG under conditions in which the apoptosis induced by VP16 or TNF was not affected. The JNK/p38 activation by EGCG was also potently inhibited by NAC, whereas those by VP16 and TNF were either not or only minimally affected by NAC. In addition, dithiothreitol also suppressed both apoptosis and JNK/p38 activation by EGCG, and EGCG-induced activation of MAP kinase kinase (MKK) 3/6, MKK4 and apoptosis-regulating kinase 1 (ASK1) was suppressed by NAC. Dominant negative ASK1, MKK6, MKK4 and JNK1 potently inhibited EGCG-induced cell death. EGCG induced an intracellular increase in reactive oxygen species and GSSG, both of which were also inhibited by NAC, and the decreased synthesis of glutathione rendered the cell susceptible to EGCG-induced apoptosis. Taken together these results strongly suggest that EGCG executed apoptotic cell death via an ASK1, MKK and JNK/p38 cascade which is triggered by NAC-sensitive intracellular oxidative events in a manner distinct from chemically induced or receptor-mediated apoptosis.

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). MATERIALS AND 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.