- Top of page
- MATERIALS AND METHODS
Granulocyte colony-stimulating factor (G-CSF)-induced alteration of phosphoprotein during differentiation of HL-60 cells was studied. From the two-dimensional gel electrophoresis analysis of phosphoproteins, a 45 kD phosphoprotein in the cytosolic fraction of DMSO-pretreated HL-60 cells was rapidly dephosphorylated by the addition of G-CSF. This 45 kD phosphoprotein migrated into four or five spots between 4.5 and 5.5 pI. The dephosphorylation of 45 kD protein was observed within at least 10 min and reached a maximum at 60 min. Phosphoamino acid analysis showed that only serine residue of 45 kD phosphoprotein was phosphorylated, suggesting that G-CSF induced an activation of serine phosphatase. Furthermore, Staurosporine and calphostin C inhibited the phosphorylation of 45 kD protein, suggesting that protein kinase C or its downstream kinase(s) is involved in the phosphorylation of 45 kD protein. These results indicate that G-CSF causes dephosphorylation of a 45 kD cytosolic phosphoprotein which may play a role in signal transduction of G-CSF.
Granulocyte colony-stimulating factor (G-CSF) is a cytokine with a potent neutropoietic activity. It was originally isolated from a human bladder carcinoma cell line 5637 ( Welte et al, 1985 ). G-CSF is considered to be a physiological haemopoietic factor implicated in neutrophil production ( Lieschke & Burgess, 1992). In vivo trials proved that exogenously injected G-CSF increased the level of the functionally mature neutrophils in the circulation ( Cohen et al, 1987 ; Tamura et al, 1987 ; Welte et al, 1987 ). G-CSF has been shown to be a positive regulator of granulopoiesis and to act at different stages of myeloid cell development ( Avalos, 1996).
Since HL-60 cells are able to differentiate into neutrophils in response to various stimulants, they have been studied as a model of granulopoiesis ( Collins et al, 1978 ; Breitman et al, 1980 ). For example, dimethyl sulphoxide (DMSO) or retinoic acid (RA) cause the differentiation of HL-60 cells into neutrophilic cells, and this differentiation is thought to be a model of neutrophilic differentiation in bone marrow. On the other hand, the neutrophilic differentiation of HL-60 cells is potentiated by G-CSF, suggesting that studying the effect of G-CSF on HL-60 cell differentiation may provide useful information concerning G-CSF-dependent regulation of neutrophil lineage cell differentiation and proliferation. It has been reported that, through the G-CSF-receptor, G-CSF enhanced the JAK-STAT pathway and raf-1-MAPK cascade ( Dong et al, 1995 ; Nicholson et al, 1995 ; Bashey et al, 1994 ) in myelogenic cells, and these signalling cascades are thought to play an important role in differentiation and proliferation. However, ser/thr phosphorylation and dephosphorylation cycles also seem to be very important in the signal transduction of cytokines ( Wen et al, 1995 ; Zhang et al, 1995 ). On the other hand, the molecular changes induced by G-CSF during neutrophilic differentiation have not been fully elucidated.
In this paper we have tried to clarify the molecular mechanism of G-CSF-dependent signal transduction in the differentiation of HL-60 cells into neutrophilic cells. Therefore we attempted to analyse G-CSF-dependent alteration of phosphoproteins in HL-60 cells during neutrophilic differentiation.
- Top of page
- MATERIALS AND METHODS
G-CSF plays an important role in both proliferation and maturation of neutrophilic-lineage cells ( Avalos, 1996). Although the G-CSF receptor belongs to the cytokine receptor superfamily, which lacks an intracellular kinase homology domain, G-CSF induces tyrosine phosphorylation of cellular proteins in myelogenic cells, resulting in the enhancement of proliferation ( Dong et al, 1995 ; Nicholson et al, 1995 ; Bashey et al, 1994 ). G-CSF induces the activation of receptor-associated protein kinases, Jak 1 and 2, which phosphorylate STAT3 ( Dong et al, 1995 ). Furthermore, tyrosine-phosphorylated STAT3 is thought to bind to a specific promoter region and cause the transcription of the gene concerned. Recently, it was reported that a kinase also phosphorylated serine residue of this transcription factor upon stimulation of cytokines ( Wen et al, 1995 ; Zhang et al, 1995 ). However, the role of phosphorylation of G-CSF action other than tyrosine phosphorylation has not been fully elucidated.
In the present study, to clarify the role of G-CSF in the differentiation of HL-60 cells, we examined the G-CSF-induced alteration of phosphoproteins in DMSO-pretreated HL-60 cells. The 45 kD phosphoprotein in the cytosolic fraction was specifically dephosphorylated by the addition of G-CSF. The G-CSF-induced dephosphorylation of the 45 kD protein was observed within 10 min after the addition of hG-CSF. Phosphoamino acid analysis indicated that the 45 kD phosphoprotein dominantly possessed a phosphoserine, indicating that the addition of hG-CSF may activate a phosphoserine phosphatase. On the other hand, staurosporine caused marked dephosphorylation of a 45 kD protein and calphostin C also caused a slight decrease in phosphorylation of a 45 kD protein, suggesting that the 45 kD protein may be phosphorylated by C-kinase or its downstream kinase.
Recently, not only kinases but also phosphatases have been reported to play important roles in many cellular activities ( Good et al, 1996 ; Tanaka et al, 1995 ). On the other hand, H-7-sensitive kinases cause the phosphorylation of the serine residue of STAT1 or STAT3 through many cytokine receptors, resulting in the enhancement of transcription of target molecules ( Wen et al, 1995 ; Zhang et al, 1995 ). The regulation of phosphorylation and dephosphorylation cycles in these processes may be very important for such a cytokine action. The G-CSF-dependent 45 kD dephosphoprotein is thought to play a role in the differentiation of HL-60 into neutrophils.
In the cytosol, we also identified a 55 kD phosphoprotein whose IEF migration patterns were very similar to those of a 45 kD phosphoprotein. The phosphorylated state of the 55 kD protein did not alter even in the presence of G-CSF. From the present data we could not determine whether or not both proteins interact or form heterodimers in physiological state. Both phosphoproteins could not be detected by either Coomassie or silver staining, indicating that these proteins are very minor components. According to Western blotting analysis using anti MAP kinase antibody, the 45 kD protein is not MAP kinase (data not shown). Furthermore, TNF-α has been shown to cause enhancement of neutrophilic differentiation in DMSO-treated HL-60 cells in term of O−2 generating activity and expression of fMLP receptor (unpublished observations). However, the addition of TNF-α did not induce the dephosphorylation of the 45 kD protein, suggesting that the dephosphorylation of the 45 kD protein is specific for G-CSF action.
Recently, okadaic acid or calyculin A, marine sponge toxins, were revealed to be potent inhibitors of phosphatase 1 and 2A ( Yano et al, 1995 ), and cause hyperphosphorylation in many cells. So, we attempted to clarify whether or not these inhibitors blocked G-CSF-dependent enhancement of HL-60 differentiation into neutrophilic cells. These phosphatase inhibitors, however, caused marked cytotoxicity in the HL-60 cells. In the present study the role of G-CSF-dependent dephosphorylation of a 45 kD protein in the differentiation of HL-60 cells into neutrophils remains unclear. However, the analysis of its role is proceeding in our group.