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Keywords:

  • G-CSF;
  • HL-60 cells;
  • dephosphorylation;
  • phosphatase;
  • neutrophilic differentiation

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

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.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

Materials

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) was kindly donated by Chugai Pharmaceutical Company Ltd, Japan. Cytochrome c (type VI), formyl methionyl-leucylphenylalanine (fMLP), and zymosan were obtained from Sigma Co. fMLP was dissolved in DMSO and stored at −20°C. Opsonized zymosan was prepared by incubation with fresh guinea-pig serum for 30 min at 37°C. Diisofluorophosphate (DFP) was from Fluka (Switzerland); 32P-labelled orthophosphate (in aqueous solution, HCl-free, carrier-free) was obtained from Amersham Co. (U.K.).

Culture of HL-60 cells

HL-60 cells were obtained from Japan Cell Resource Bank (Tokyo, Japan). Frozen stock cells were thawed and cultured in RPMI 1640 containing 10% fetal calf serum under 5% CO2. In order that the cell supply should be of the same quality in the series of experiments, frozen cells from the same lot were thawed every 3 months and used for experiments. For the differentiation of HL-60 cells into neutrophil-like cells, exponentially growing cells, with a doubling time of about 40 h, were collected and usually resuspended in 1.25% DMSO. At 2–3 d after the addition of DMSO, various amounts of rhG-CSF were added to the cell suspensions, and re-incubated for a subsequent 4–5 d.

Protein phosphorylation in HL-60 cells

After incubation with DMSO for 2–3 d, HL-60 cells were collected and washed with phosphate-free RPMI 1640 medium. The cells (1 × 107 cells/ml) were re-suspended in phosphate-free RPMI 1640 containing 5% dialysed fetal calf serum, and then incubated with 18.5 MBq of [32P]orthophosphate/ml for 2 h at 37°C. At the end of the incubation period, various amounts of rhG-CSF were added to the cell suspension and subsequently incubated at 37°C. After incubation for the indicated periods, the cell suspension was mixed with chilled phosphate-buffered saline containing phosphatase inhibitors and protease inhibitors ( Hayakawa et al, 1986 ). After separation of the cytosolic and membrane fractions, two-dimensional gel electrophoresis was performed. The details of the experimental conditions were the same as described ( Hayakawa et al, 1986 ). The 32P incorporated to a protein spot was determined by autoradiography by using X-ray film or radioluminography using a Bioimage Analyser (Fuji Film Co., Tokyo, Japan).

Electroblotting and determination of phosphoamino acid

After two-dimensional electrophoresis was performed as above, the proteins separated on the gels were electrophoretically transferred to PVDF membranes at 2 mA/cm2 for 2 h. The spots of the 45 kD phosphoprotein, which were identified by autoradiography, were excised from the blots. The 45 kD phosphoprotein was hydrolysed in HCl vapour at 110°C for 2 h. Then, the membranes were wetted with 20 μl of methanol, and extracted with 200 μl of distilled water. The extract was dried in an Eppendorf tube by Speed Vac and solubilized in 20 μl of 3 m M cold phosphoamino acid mixture solution containing orange G (electrophoresis marker). The electrophoresis was performed as described previously ( Suzuki et al, 1995 ). The position of standard phosphoamino acids were identified by ninhydrin staining.

Superoxide generation by differentiated HL-60 cells

The superoxide generation by differentiated HL-60 cells was continuously measured at 37°C by recording the reduction of cytochrome c ( Yamaguchi et al, 1986 ). The basal assay mixture contained 1 × 106 cells, 50 μM cytochrome c, 5 m M glucose, and 0.5 m M CaCl2 in 1 ml of HEPES-buffered saline. Cells were activated with 500 n M fMLP or 1.25 mg/ml opsonized zymosan.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

Effects of rhG-CSF on the differentiation of HL-60 cells

Neutrophilic differentiation of HL-60 cells pretreated with DMSO was augmented by rh G-CSF in terms of O2 generating activity (Fig 1), as reported previously ( Bollag, 1991; Sakashita et al, 1991 ). Since G-CSF alone failed to initiate the differentiation of HL-60, the role of G-CSF on HL-60 cells may be considered as a potentiator for neutrophilic differentiation.

image

Figure 1. .25% DMSO. 2 d after the addition of DMSO, 60 ng/ml G-CSF was added to DMSO-treated HL-60 cells, and cells were subsequently incubated with or without G-CSF for 5 d. The O2 generation by differentiated cells was initiated with 0.5 μM fMLP.

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Next, we examined whether hG-CSF caused the alteration of phosphoprotein in the differentiating HL-60 cells. As shown in Fig 2, G-CSF caused the dephosphorylation of 45 kD protein in the cytosolic fraction. This 45 kD phosphoprotein in the absence of G-CSF migrated into four or five spots between 4.5 and 5.5 of pI values. Upon addition of hG-CSF, radioactive intensities of all these spots were reduced. Except for the 45 kD protein, there were no changes in phosphoprotein patterns between G-CSF-treated and untreated HL-60 cells. Interestingly, we also identified the presence of 55 kD phosphoproteins whose migration pattern in the first IEF dimension was very similar to that of 45 kD phosphoprotein. However, the phosphorylation state of 55 kD phosphoprotein was not altered by the presence or absence of hG-CSF. On the other hand, in the plasma membrane fraction there was no significant change of phosphoprotein pattern in response to G-CSF (data not shown).

image

Figure 2. D-gel electrophoresis and phosphoproteins were autoradiographically visualized. Arrow heads indicate the 45 kD phosphoproteins.

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In order to characterize 45 kD phosphoprotein, we examined the kinetics of dephosphorylation of this protein after the addition of G-CSF. As shown in Fig 4, the 45 kD protein was dephosphorylated by the addition of G-CSF within 10 min, and the dephosphorylation of the 45 kD protein reached a maximum at 60 min. For the analysis of phosphoamino acid of the 45 kD phosphoprotein, the bands of the 45 kD phosphoprotein prepared from DMSO-treated HL-60 cells were excised and hydrolysed in 6 N HCl. The resulting phosphoamino acid pattern clearly shows that the 45 kD phosphoprotein dominantly possessed phosphoserine but not phosphotyrosine nor phosphothreonine (Fig 5). By using protein kinase inhibitors we investigated which kinase was involved in the phosphorylation of the 45 kD protein (Fig 3). Staurosporine (0.1 μM) markedly inhibited the phosphorylation of the 45 kD protein and calphostin C, a potent C-kinase inhibitor, also caused a decrease in the phosphorylation of the 45 kD protein, whereas herbimycin A did not alter the phosphorylation state of the 45 kD protein. These findings suggest that C-kinase or its downstream kinase(s) was involved in the phosphorylation of the 45 kD protein.

image

Figure 4a. 5 kD protein induced by G-CSF over 180 min.

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image

Figure 5.  kD protein was excised and hydrolysed under HCl vapour. After the separation of phosphoamino acids, each spot of phosphoamino acid was visualized by a BAS-2000 Imageplate analyser.

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image

Figure 3. 2P-loaded HL-60 cells were incubated for 10 min with 1 μM calphostin C, 0.1 μM staurosporine, 1.5 μM herbimycin A or DMSO alone. After incubation, the cytosol fraction was prepared from these cells. Arrowheads indicate the 45 kD phosphoproteins.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

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 O2 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.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

We thank Japanese Cancer Research Resources Bank for the kind supply of HL-60 cells. This work was supported in part by grant-in-aid from Japan Health Sciences Foundation.

References

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References
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