MAP kinase upregulation after hematopoietic differentiation: role of chemotaxis

Jason A. Lehman1, Cassandra C. Paul2,3, Michael A. Baumann2,3, and Julian Gómez-Cambronero1

Departments of 1 Physiology and Biophysics and 2 Medicine, Wright State University School of Medicine, Dayton 45435; and 3 Research Service, Dayton Veterans Affairs Medical Center, Dayton, Ohio 45428


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Mitogen-activated protein kinase (MAPK) isoform p42 is known to be active in exponentially growing cells at several points of the cell cycle. A high basal activity was present in three cell lines representative of immature myeloid cells tested: uHL-60, AML-14, and MPD. However, DMSO-induced differentiation of HL-60 cells (dHL-60) and subsequent expression of the neutrophilic phenotype occurred with a concomitant reduction on the basal level of MAPK activity. Simultaneously, extracellular stimuli like the cytokine granulocyte/macrophage colony-stimulating factor (GM-CSF) induced a fast (<10 min) and robust response. In terms of MAPK activity, the more mature the cell was, the higher the corresponding activity, in the three differentiation series considered: AML-14 < 3D10; MPD < G-MPD; uHL-60 < dHL-60 < neutrophils. Interestingly, peripheral blood neutrophils expressed the highest (16-fold) MAPK activation level in response to GM-CSF. Finally, using the specific MAPK inhibitor PD-98059, we demonstrated that MAPK activation is needed for neutrophil chemotaxis toward interleukin-8 and its priming by GM-CSF. Since neutrophils are terminally differentiated cells, GM-CSF does not serve a purpose in proliferation, and it must trigger the recruitment of selective signal transduction pathways particular to that final stage that includes enhanced physiological functions such as chemotaxis.

mitogen-activated protein kinase; granulocyte/macrophage colony-stimulating factor; neutrophils; cell differentiation; leukemic cells


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PROLIFERATION AND DIFFERENTIATION of myeloid progenitors is regulated by a family of hematopoietic growth factors, including granulocyte/macrophage colony-stimulating factor (GM-CSF). GM-CSF stimulates DNA synthesis and transcription in immature myelo-monocytic progenitor cells and acts on mature macrophages and granulocytes by priming them for their phagocytic functions. Specifically, at the site of infection or inflammation, GM-CSF enhances phagocytosis of invading pathogens by granulocytes and macrophages by increasing degranulation, adherence, alkalization of the cytosol, inhibition of migration, and the release of superoxide anions, platelet-activating factor, and arachidonic acid (reviewed in Ref. 12).

GM-CSF is one of the hematopoietic growth factors required for the proliferation and differentiation of myeloid-committed normal progenitor cells, but it fails to elicit differentiation in most transformed cells. To achieve differentiation in vitro, additional induction with an agent such as DMSO is needed (26). The mechanism by which DMSO induces maturation is still largely unknown. It might induce the acquisition of effector functions that are characteristic of mature neutrophils, such as key signal transduction molecule links (4, 19, 25).

The postreceptor signaling pathways utilized by GM-CSF are the subject of intense study (1, 2, 5, 9, 11, 31). One of the earliest events is tyrosine phosphorylation of the receptor beta -subunit (GM-CSF · Rbeta ). Some important downstream events are 1) activation of the Ras (MAPK/ERK2) mitogen-activated protein kinase/extracellularly regulated kinase 2 pathway, a common signaling route for many hematopoietic growth factors, 2) tyrosine phosphorylation and activation of p38 MAPK, an important crosspoint in neutrophil signaling (18), and 3) activation of p42 MAPK that leads to Ser/Thr phosphorylation of one of its substrates, p90rsk (8), known to be essential for cytostatic factor arrest (3). In the case of hematopoietic models, it is well known that the expression and states of various signaling molecules, such as src-like kinases, change with myelo-monocytic differentiation. Thus there is reason to anticipate that receptors may effect different signals depending on the effector molecules downstream of them that depend on the differentiation state of the cell.

This study addresses the cell signaling pathways that underlie the uniqueness of the neutrophil's functionality, compared with well-established cycling cells. Although GM-CSF has been shown from its discovery as being needed for DNA synthesis in progenitor cells, the focus of the present work is on how hematopoietic cells respond to the cytokine at several stages of differentiation. We provide evidence showing that neutrophils could have an enhanced functionality (such as chemotaxis) when primed by the cytokine as it occurs during the course of an infection. Upregulation of p42 MAPK might be at the crux of these events.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Materials and antibodies. The promyelocytic leukemic (M2 myeloblast-derived) HL-60 cell line was from American Type Culture Collection (Rockville, MD); the acute myeloid leukemia cell line AML-14 and the more differentiated (eosinophilic) subline AML-14.3D10, as well as the MPD cell line (non-chronic myelogenous leukemia myeloproliferative disorder) were developed by the authors (21, 22); tissue culture material was from Becton Dickinson (Oxnard, CA) and Costar (Cambridge, MA); GM-CSF and granulocyte colony-stimulating factor (G-CSF) were from R&D Systems (Minneapolis, MN); cAMP-dependent protein kinase inhibitor, calphostin, anti-rabbit IgG (agarose beads), differentiation kit, trypan blue, nitro blue tetrazolium (NBT) reduction kit, and myelin basic protein (MBP) were from Sigma (St. Louis, MO); electrophoresis chemicals were from Bio-Rad Laboratories (Richmond, CA); [gamma -32P]ATP (30 Ci/mmol) was from Amersham (Arlington Heights, IL); PD-98059 was from BioMol (Plymouth Meeting, PA); ion-exchange chromatography cellulose phosphate paper was from Whatman (Hillsboro, OR); antiphosphotyrosine (anti-PY; PY20 clone), anti-p42mapk, and anti-p90rsk kinase antibodies, MAPK peptide substrate APRTPGGRR, and ribosomal S6 kinase (RSK) peptide substrate RRRLSSLRA were from Upstate Biotechnology (Lake Placid, NY).

Isolation of peripheral blood neutrophils. The isolation procedure was based on English and Andersen (7). Between 50 and 55 ml of blood were collected from the antecubital veins of healthy individuals (who signed an Institutional Review Board-approved consent form), using sodium citrate as anticoagulant. Blood was mixed with 15 ml of 6% dextran, allowed to settle, and the plasma and buffy coat were removed and spun down at 800 g for 5 min. The pellet was resuspended in 35 ml of saline and centrifuged again for 15 min at 10°C in a Ficoll-Histopaque discontinuing gradient. Neutrophils were recovered, and contaminating erythrocytes were lysed by hypotonic shock. Cells were washed, and the pellet was resuspended in Hanks' balanced salt solution (HBSS). No neutrophil aggregation (i.e., the hallmark for neutrophil activation) was observed by using this isolation procedure. Viability was >98% as per trypan blue exclusion.

Leukemic cell culture and induction of differentiation. HL-60, AML-14 and its subline AML-14.3D10, and pluripotent MPD cells were grown at 37°C in a 5% CO2 incubator in RPMI 1640 medium supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum, 2 mM L-glutamine, and 5 µg/ml gentamicin. Stock cultures were diluted with fresh medium every other day, with extra medium on weekends, so that cell density was maintained between 0.1-1.0 × 106/ml. Vented culture flasks were changed biweekly. Cultures were checked for mycoplasma contamination once a month. Cell extracts were prepared as indicated in the next section. HL-60 cells were induced to differentiate by incubation with 1.25% (vol/vol) DMSO for a maximum of 6 days (19), but normally 3-4 days sufficed to achieve the neutrophilic phenotype. Aliquots were sampled daily and assessed for differentiation. To induce differentiation of the MPD cell line to mature neutrophils, cells were grown at 37°C in a 5% CO2 incubator in RPMI 1640 medium continuously supplemented with 20 pM G-CSF for 5 days. Viability assays were routinely conducted with 0.4% trypan blue stain in cell preparations before all analyses. Average counts were uHL-60 = 1.8 × 106 cells/ml (viability ~95%); 4-day DMSO: dHL-60 = 1.4 × 106 cells/ml (viability ~93%); MPD = 1.2 × 106 cells/ml (viability ~95%); 5-day G-CSF: G-MPD = 1.6 × 106 cells/ml (viability ~92%).

Assessment of differentiation. In standard procedures with HL-60, at 80 h of DMSO incubation, ~75% of the cells matured to at least the myelocytic stage and acquired the ability to release superoxide anion in response to bacterial extract challenge. To ascertain that DMSO produced the desired neutrophilic phenotype in HL-60 cells, cells were assayed for 1) morphology of Wright-stained slides; 2) cytopreparations of NBT-treated preparations of cells stimulated with 5 µl of nonviable bacterial extracts. Positive cells contained formazan deposits as dark, irregularly shaped crystal inclusions in the cytoplasm and were (positive/total) 2/290 = 0.6% (uHL-60); 22/227 = 9.7% (dHL-60); and 137/468 = 29% (neutrophils); 3) flow cytometric analysis of surface expression of differentiation-related antigens was performed with fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody against CD11b, CD11c, CD13, CD14, CD16, or CD54.

Preparation of cell extracts, immunoprecipitation, and Western blotting. Cells were washed in HBSS (to eliminate any potential autocrine cytokine production) and incubated with GM-CSF at 37°C. Aliquots were spun down for 30 s at 7,000 g, and pellets resuspended in 200 µl lysis buffer (12 mM Tris · HCl, pH 7.2, 0.75 mM NaCl, 100 µM sodium orthovanadate, 10 µM phenylmethylsulfonyl fluoride, 5 µg/ml each of aprotinin, pepstatin A, and leupeptin, and 0.12% Triton X-100) and sonicated on ice. Total cell lysates were centrifuged (for 2 min at 14,000 g at 4°C), and supernatants (~1 mg/ml protein) were saved for immunoprecipitation (16). Immune complexes were resuspended in a final volume of 60 µl with lysis buffer and mixed with 2× SDS sample buffer (1:1 vol/vol) for subsequent Western blotting or with kinase buffer for subsequent kinase assays. When required, bands from X-ray films derived from Western blots were scanned and area x optical density was calculated. When mobility shift measurements were required, the distance migrated by a particular band in Western blots was measured (in mm) and extrapolated in a SDS-polyacrylamide gel calibration curve for known molecular weight protein standards vs. distance.

In vitro kinase assays. The enzymatic activities in whole lysates or immune complexes were measured as described (23) with some modifications. The phosphoacceptor peptide, 1 mM APRTPGGRR [bearing the phosphorylation site of amino acids 95-98 (PRTP) from bovine MBP], was diluted in freshly prepared kinase buffer (13.4 mM HEPES, pH 7.3, 25 mM MgCl2, 30 µM Na2VO2, 5 µM p-nitrophenyl phosphate, 2 mM EDTA, 2 µM cAMP-dependent kinase inhibitor, 5 µM calphostin, 21 µCi [gamma -32P]ATP/ml, and 68 mM unlabeled ATP). The phosphotransferase reaction was initiated by mixing aliquots (20 µl) of kinase buffer with the cell lysates or immunoprecipitates at 1:1 (vol/vol) ratio for 20 min and was terminated by blotting 20 µl of the reaction mixture onto cellulose phosphate P81 papers. These were dried and counted for radioactivity. Controls were run in parallel, by which the reaction was carried over with all the indicated reagents in the absence of the phosphoacceptor peptide and radioactivity counts were subtracted from the experimental conditions.

In-gel renatured kinase assays. MAPK activity was also measured by an "in-gel" kinase assay, as described in (17), with some modifications. Briefly, 250 µg/ml MBP, to measure p42mapk, or 100 µg/ml RSK peptide (RRRLSSLRA = amino acids 231-239 of human protein S6), to measure p90rsk, was mixed with acrylamide in an SDS-PAGE gel (1.5 mm thin). After electrophoresis, gels were freed of SDS by incubation with 20% isopropanol in buffer A (100 mM Tris · HCl, pH 8, and 5 mM 2-mercaptoethanol), then with buffer A containing 6 M guanidine-HCl, and finally, in buffer A containing 0.04% Tween 40 at 4°C for 6 h. Renatured gels were overlaid with kinase buffer (20 mM Tris · HCl, pH 8, 10 mM MgCl2, and 25 mCi [gamma -32P]ATP) and incubated at room temperature for 30 min. Gels were washed with 1% pyrophosphate in 5% TCA, dried, and exposed to X-ray films. For the immunocomplex kinase assay, agarose beads were resuspended in 30 µl of lysis buffer, boiled for 5 min, spun down (15 s, 14,000 g), and supernatants were loaded in the substrate-embedded SDS gels. When needed, the kinase of interest was excised off the dried gel and counted for radioactivity.

Functional assay: chemotaxis. Neutrophils (5 × 105) in chemotaxis buffer (HBSS + 1 mM CaCl2, 1 mM MgCl2, and 0.1% BSA) were placed on the upper chambers of 6.5-mm Transwell dishes (Costar) that were separated from the lower chambers by a 5-µm pore nucleopore polycarbonate membrane. Interleukin-8 (IL-8) was added in 0.6 ml of chemotaxis buffer to the lower chamber. The dishes were incubated for 1 h at 37°C under a 5% CO2 atmosphere, and the cells that migrated to the lower chambers were counted on a microscope by using a hemocytometer. Viability at the end of the assay in both chambers remained >97 ± 2%, even in the presence of MAP or ERK kinase (MEK) inhibitor, ruling out a toxic effect. Maintenance of the experiment for 2 h proportionally increased the number of migrating cells, but viability decreased to >75 ± 3%.

Statistical analysis. The difference between means was assessed by the single factor analysis of variance test. Probability (P) of <0.05 was considered to indicate a significant difference. Several P values are given in the figure legends.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The extent of MAPK tyrosyl phosphorylation in response to GM-CSF is greater in mature than in undifferentiated leukemic cells. The expression of surface antigens in both undifferentiated (uHL-60) and differentiated HL-60 (dHL-60) cells was first investigated to confirm that DMSO had induced cell maturation. Upregulation was observed in nearly all surface antigens studied by flow cytometry (Fig. 1). Noticeable changes in CD11b (14, 27) and CD13 (zinc metalloproteinase) were present, and equivalent levels were expressed in both progenitors and mature myelomonocytic cells as expected. The assayed integrins, intercellular adhesion molecules, immunoglobulin fragment Fc, and lipopolysaccharide (LPS) receptor, as representatives of granulocyte extravasation, phagocytosis, and antibody-dependent cell-mediated cytotoxicity, confirmed the presence of the neutrophilic phenotype in dHL-60 cells.


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Fig. 1.   Cell surface expression of differentiation antigens on HL-60 cells. Samples (100 µl) of 1 × 104 cells were incubated with IgG2b antibodies against several antigens and counted by flow cytometry. A: exponentially growing, undifferentiated HL-60 cells (uHL-60). B: differentiated HL-60 cells by 3-day induction culture with 1.25% DMSO (dHL-60, 3 days). Antigens are split in 2 panels for the sake of clarity: CD11b, CD16, and CD14 (left, A and B); CD11c, CD13, and CD54 (right, A and B). X-axes scales are logarithmic. Shaded areas represent background staining.

When uHL60 were acutely (3-15 min) stimulated with GM-CSF, an increase in phosphotyrosine was observed as a function of the stimulation time, as demonstrated in anti-PY blots of whole cell lysates (Fig. 2A) and in anti-PY blots of anti-p42mapk immunocomplexes (Fig. 2B) with similar protein loading. However, when the same cells that had been induced to mature with continuous exposure to DMSO (dHL-60) were stimulated with GM-CSF, the extent of the response was greatly augmented (Fig. 2, A and B, middle). Furthermore, neutrophils isolated from peripheral blood were also subjected to treatment with GM-CSF in conditions similar to both uHL-60 and dHL-60 experiments, and it was observed that the extent of the response was the greatest of the three (Fig. 2, A and B, bottom). Qualitatively, the activity peaked at 5 min in uHL-60 and at 10 min in dHL-60 and still remained in plateau at 15 min in neutrophils. The extent of MAPK phosphorylation in response to GM-CSF stimulation from the PY band in immunoprecipitates was uHL-60 < dHL-60 < neutrophils (Fig. 2C).


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Fig. 2.   Effect of granulocyte/macrophage colony-stimulating factor (GM-CSF) on mitogen-activated protein kinase (MAPK) tyrosine phosphorylation on leukemic and neutrophil cells. Either freshly cultured HL-60 (uHL-60), HL-60 induced to express the neutrophilic phenotype by continuous exposure of 1.25% DMSO for 3-4 days (dHL-60), or peripheral blood neutrophils (Neut) were resuspended at the density of 1 × 107 cells/ml and incubated at 37°C with 270 pM GM-CSF for the indicated lengths of time. A: Western blots (WB) were prepared from whole cell lysates and probed with anti-PY antibodies and subsequently with anti-p42mapk antibodies, after stripping the first one (alpha -PY). B: anti-p42mapk immunoprecipitation (IP) in cell extracts prepared in the presence of boiling 1% SDS and 10 mM Tris, pH 7.4. Resulting blots from immunocomplex agarose beads were probed with either anti-PY or anti-p42mapk antibodies. C: optical density (OD) and area scans were performed on the PY band of immunoprecipitates relative to MAPK shown in B. Data are representative of 4 separate experiments.

Since MAPK is usually phosphorylated on both tyrosine and threonine residues for full enzymatic activity, additional studies were performed to analyze threonine phosphorylation of MAPK with an anti-TEY antibody, and they confirmed the same pattern of activation (not shown). Also, as indicated in Fig. 3, p42mapk phosphorylation was dependent on the dose of GM-CSF. Results in this graph indicate that 1) basal MAPK total mass protein diminished as maturation progressed, with the least in neutrophils; and 2) phosphorylation of MAPK, in response to GM-CSF, increased as maturation progressed, with a maximum in neutrophils. Scans performed on the upper band resulted in sigmoidal curves (Fig. 3C) from which the EC50 was lowest in neutrophils. Time-course and dose-response experiments both provided evidence for fundamental differences (both quantitative and qualitative) in GM-CSF functional signaling with cell maturation.


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Fig. 3.   Effect of GM-CSF on MAPK tyrosine phosphorylation on hematopoietic cells: dose response. Leukemic and neutrophil cells at the density of 1 × 107 cells/ml were incubated at 37°C for 5 min with the indicated concentrations of GM-CSF. A: Western blots were prepared from whole cell lysates and probed with anti-p42mapk antibodies. B: the same blots stripped of the antibody and reprobed with anti-PY. Shown are regions of the blots that span from 35-50 kDa. C: mobility shifts quantitation in the 3 different cell types. Shown are the increments (in area × OD) of the phospho-MAPK band (upper band in doublet) after exposure to the different doses of GM-CSF. , uHL-60; , dHL-60; , neutrophils. Shown are results from 3 independent experiments.

To see if the above results were particular for HL-60 or were extensive to other leukemic cells, similar analysis was extended to the MPD cell line that closely resembled a CD34+ stem cell in that it was pluripotent (21). This cell line consisted of 60-70% immature population and 30% mature, of which 80-90% were neutrophils (MPD = undifferentiated). By continuous exposure of the cells to the cytokine G-CSF for 3-5 days, up to 50% of cells appeared to be mature neutrophils (G-MPD = differentiated). Both MPD and G-MPD cells were assayed for their ability to engage in MAPK activation in response to a brief GM-CSF challenge. Figure 4, A and B, indicates that tyrosine phosphorylation, in response to short stimulation with GM-CSF, was greater in the mature population compared with the less differentiated cells.


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Fig. 4.   MAPK activity in the non-chronic myelogenous leukemia myeloproliferative disorder (MPD) cell line in response to GM-CSF stimulation. A: immunoprecipitation with anti-p42mapk and Western blot with anti-PY antibodies from immature (MPD) and differentiated (G-MPD) cells. B: densitometry of p42-phosphotyrosine bands expressed as area × OD (arbitrary units). Shown is the mean ± SE of 3 independent experiments. open circle , MPD cells; , G-MPD cells.

The highest MAPK enzymatic activity after stimulation occurs in neutrophils while differentiated leukemic cells have an intermediary activity. Results from immunoblots and immunoprecipitates are indicative of a conformational change of the protein due to phosphorylation, and consequently, an increase in enzymatic activity could be expected. To fully assess MAPK enzymatic activity with confidence, we used all contemporary methodology that exists; e.g., Western blotting and study of shift mobility, in vitro 32P[ATP] incorporation, immunokinase assay with specific immunocomplex preparations, and in-gel renatured kinase assay in precleared/immunoprecipitated samples. The first approach was done initially by detecting in vitro 32P incorporation to the specific MAPK substrate APRTPGGRR. Cell lysates were prepared from an array of myeloid leukemic cells, both undifferentiated and induced to mature, including the eosinophilic cell line AML-14 and its relatively more mature subline AML-14.3D10, uHL-60, dHL-60, and HL-60 treated with all-trans-retinoic acid (aHL-60) (Fig. 5). Two points were noted. 1) The most immature cells (AML-14, MPD, and 3D10) had the highest basal level of kinase activity. This was also true for fresh, exponentially growing uHL-60 cells, in agreement with Okuda et al. (20), in that transformed, immortalized cultured cells had a high operating level of the Ras/MAPK pathway, even in the absence of growth factors. 2) Basal level was increased in response to GM-CSF stimulation, but at very different rates in the series considered. The incorporation of 32P into the phosphoacceptor substrate followed the order AML-14 approx  MPD < 3D10 approx  uHL-60 < dHL-60 approx  aHL-60 < G-MPD < neutrophils.


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Fig. 5.   In vitro MAPK activity during differentiation of leukemic cells at different stages of maturation. Kinase activity was measured in uHL-60, DMSO-treated (dHL-60), or ATRA-treated (aHL-60) HL-60 cells, mature polymorphonuclear neutrophils from peripheral circulation (Neut), AML14, or AML14.3D10 (3D10), in the absence (solid bars) or in the presence of 270 pM GM-CSF (open bars) at 37°C for 5 min. Cell lysates were prepared, and kinase activity was assayed in vitro against the MAPK substrate peptide APRTPGGRR using the same total protein concentration (20 µg in 40 µl, final reaction volume, per each condition). The figure represents the mean ± SE of 4 experiments. Significance values, *P < 0.5; #P < 0.1; @P < 0.01 compared with control in each pair.

The measurement of enzymatic activity was also done in-gel. As shown in Fig. 6A, a rapid (<= 5 min) increase in MAPK activity occurred in all cells tested, but to a greater extent in the more mature populations, with a maximum of up to 14-fold over controls in peripheral blood circulating neutrophils. Notice also that, in addition to MAPK, two additional proteins used MBP as substrate (myelin basic protein kinases, or MBPK) incorporating radioactive phosphate in the in-gel assay. These kinases were p55 MBPK and p65 MBPK. However, contrary to the p42 MAPK data, their phosphorylating activity did not change with the addition of GM-CSF, a result that was confirmed when bands were excised from the gels and counted for radioactivity (Fig. 6, B-D). To demonstrate that the p42 MBP kinase was the involved member of the MAPK family, immunoprecipitation with anti-p42mapk antibodies followed by in-gel kinase assay of the immunocomplexes confirmed that the band under study was indeed p42 MAPK and that all other kinases were absent in unstimulated cells (Fig. 7). Again, an extensive response to GM-CSF in MAPK activity was observed in the more differentiated population of HL-60 cells.


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Fig. 6.   "In-gel" MAP kinase enzymatic activity. A: lysates containing ~160 µg protein from cells unstimulated (-) or stimulated with 270 pM GM-CSF for 5 min (+) were loaded on each gel lane of SDS-gels previously copolymerized with 250 µg/ml myelin basic protein (MBP). The kinase reaction was performed by overlaying [gamma -32P]ATP on the renatured gels as indicated in MATERIALS AND METHODS. Gels were extensively washed, dried, and exposed to X-ray films to localize protein bands. The figure shows the result of a typical assay. The arrows (right) mark the position of MBP-utilizing kinases. B-D: detection of MAPK activity by liquid spectrometry. 32P radioactivity on immunoreactive gel slices were counted in a beta counter. Radioactivity associated with the p42- (B), p55- (C), and p65- (D) MBP kinases are shown in solid bars (controls) and open bars (GM-CSF-treated cells) as the mean ± SE of 3 independent experiments.



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Fig. 7.   Immunocomplex kinase assay analysis of MAPK. After immunoprecipitation with monoclonal anti-p42mapk antibody of HL-60 cell lysates, immunocomplex beads were loaded in an MBP-embedded SDS-gel, and the in-gel kinase assay was performed. The arrow (right) marks the position of p42-MAPK. Shown is a representative result out of 4 separate experiments.

p90rsk Activation follows the p42 MAPK pattern in HL-60 cells. We next studied the response of one of the MAPK substrates, the ribosomal p90rsk, to GM-CSF challenge. The presence of p90rsk was detected in leukemic cell lines with the antibody recognizing a band of the proper molecular mass (i.e., 88-90 kDa) (Fig. 8). As with MAPK, GM-CSF caused a shift in relative electrophoretic mobility of p90rsk higher in dHL-60 than in uHL-60 cells. In blots such as the one presented in Fig. 6, mass of p90rsk was measured. The presence of phosphorylation was confirmed by an in-gel renatured kinase method (Fig. 8B). Because an increase in band intensity correlated with an elevated enzymatic activity of the kinase, p90rsk from dHL-60 cells clearly responded to GM-CSF stimulation better than uHL-60. Finally, the effect was dependent on the dose (Fig. 8C) being more pronounced in dHL-60/neutrophils than in uHL-60. The pattern for p90rsk activation was uHL-60 < dHL-60 approx  Neut (neutrophils not being higher than dHL-60, as indicated in Ref. 16).


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Fig. 8.   Effect of GM-CSF on p90rsk in HL-60 cells. A: HL-60 cells were incubated at 37°C with 270 pM GM-CSF for the indicated lengths of time. Western blots were probed with anti-p90rsk antibody. Shown are regions of the blots spanning between 80 and 100 kDa. B: In-gel ribosomal S6 kinase (RSK) enzymatic activation by GM-CSF of lysates run in SDS-gels copolymerized with 100 µg/ml RSK peptide substrate (RRRLSSLRA). C: effect of GM-CSF concentration on RSK p90rsk kinase mobility shifts. Shown are the increments in kilodaltons of the 90-kDa band after exposure to the different doses of GM-CSF (inset: u = uHL-60; d = dHL-60; n = neutrophils). Symbols: , uHL-60; , dHL-60; , neutrophils. Shown are results from at least 3 independent experiments.

MAPK upregulation is related to cell function in neutrophils. Results from the above experiments suggested a blockage in the transduction of signals between the binding of GM-CSF and the activation of the kinase in uHL-60 cells but upregulation in neutrophils. To ascertain a physiological implication of this effect, we looked at chemotaxis toward a well-defined and potent neutrophil stimulus, IL-8. Cell migration toward 50 or 500 nM IL-8 was inhibited by the MEK inhibitor PD-98059 in a dose-dependent manner (Fig. 9A), with an IC50 of ~10 µM. Additionally, the role of GM-CSF for MAPK in more mature cells was underlined by the finding that chemotaxis was augmented by GM-CSF preincubation. This priming effect was also abrogated by PD-98059 (Fig. 9B).


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Fig. 9.   The dependence of MAPK on a chemotaxis functional assay. A: neutrophils were resuspended in chemotaxis buffer (see MATERIALS AND METHODS) and preincubated with the indicated concentrations of the MAP or ERK kinase (MEK) inhibitor PD-98059 for 20 min at 37°C and placed on the upper chambers of 6.5-mm Transwell dishes at a density of 5 × 105/well, in the presence or absence of interleukin-8 (IL-8; 50 or 500 nM), kept in the lower chamber. The dishes were incubated for 1 h at 37°C, and the cells that migrated were counted on a microscope using a hemocytometer. Shown is the mean of 2 independent experiments in duplicate. B: same protocol as before, except that neutrophils were primed with 1 nM GM-CSF for 30 min at 37°C before being mixed with the MEK inhibitor PD-98059 (10 or 25 µM) and subsequent IL-8 treatment (50 nM).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We show here that MAPK activation in response to GM-CSF increases with the maturational state of the cells. The ability of GM-CSF to induce tyrosine phosphorylation and activation of the p42 isoform of MAPK was studied in three well-defined cell line models and found to directly correlate with maturation stage. Even though the isoform p42mapk was present (and at a high basal level) in exponentially growing, undifferentiated HL-60 cells, only a marginal increase in tyrosine phosphorylation and enzyme activity was observed on cell stimulation with GM-CSF. A maximal response was seen in mature neutrophils. These results were replicated in other GM-CSF receptor-expressing cell line models: the blastlike cell line AML-14, its more differentiated (expressing the eosinophil phenotype) subline AML-14.3D10, and the recently described pluripotent MPD cell line (22), representative of non-CML MPD that differentiates into mature granulocytes with continuous exposure to G-CSF. Quantitatively, the order of MAPK activation was AML-14 approx  MPD < 3D10 approx  uHL-60 < dHL-60 approx  aHL-60 < G-MPD < neutrophils.

A higher phosphorylation activity in the more differentiated cells is in agreement with previous studies demonstrating p93fes and p60src (30), tyrosine phosphorylation (26), and MAP2 kinase (24) activation. Recently, a low response to GM-CSF was linked to low Janus kinase 2 levels in HL-60 cells, although this could not be extrapolated to CD34+ cells (29). In the present study, we observed that in spite of MAPK being maximally activated by GM-CSF in neutrophils, one of its main substrates, p90rsk, reached its maximum of activation only in dHL-60 cells. This indicates that pathways other that the classical ones leading to cell division must exist, especially for terminally differentiated cells.

While in stem, cycling cells, MAPK is important for the exit G0 right-arrow G1 and for the spindle formation; in mature granulocytes, a massive response of MAPK to short stimulation by GM-CSF must have a different functionality. This stimulation can still be related to microtubule assembly. Precisely, the first name proposed for MAPK was "microtubule-associated protein kinase" for its ability to phosphorylate microtubule-associated proteins, or MAPs. It is likely that in neutrophils, an activation of these or related proteins can lead to increased cell movement events in the infection site, namely, chemotaxis and phagolysosome formation. Our results (Fig. 9) point to the direction of MAPK being important in neutrophil chemotaxis and the role of GM-CSF for MAPK activation in more mature cells. Still, other pathways could act in parallel. Thus, while the activation of the MAPK pathway is necessary for insulin-like growth factor-I-induced mitogenesis, the activation of phosphatidylinositol 3-kinases (PI 3-kinases) is very important for cell migration and integrin activation (15). cAMP-induced activation of the sodium taurocholate cotransport may be mediated via the PI 3-kinases/protein kinase B signaling pathway requiring intact actin filaments (28). Also, and equally important, the differential effect of stimuli in a particular cell type should be kept in mind. For example, GM-CSF, LPS, or tumor necrosis factor-alpha (TNF-alpha ) stimulate cellular functions that are not induced by phorbol 12-myristate 13-acetate or ionomycin (32).

A role for the "classic" MAPK (ERK2, p42mapk) in the cell cycle has been recently demonstrated (10, 13). MAPK is a key player in cycling cells. Its activity was elevated in mitosis of HeLa cells, but only in interphase did cells respond to epidermal growth factor, as if mitotic cells were sheltered from perturbing signals from the exterior. As differentiation reaches a terminal phase, the cyclic pattern changes to a steady state in which MAPK is available for activation only when cells engage in their physiological microtubule-related functions, migration and phagocytosis, both needed in the inflammation site and primed by cytokines such as GM-CSF.

Thus the presence of a highly stimulatable MAPK activity in neutrophils is not by any means a leftover of mitotic, hematopoietic stem cells from which neutrophils derive. Rather, this enzyme might be regulated differently and uniquely. We are currently continuing our work in the direction of finding the precise connection between the two cell signaling pathways presented herein and the motility-related neutrophil functionality.


    ACKNOWLEDGEMENTS

We thank Dr. Peter K. Lauf for critical reading of this manuscript and helpful suggestions.


    FOOTNOTES

This work was supported in part by National Heart, Lung, and Blood Institute Grant HL-056653 and American Heart Association Grant 9806283 (to J. Gómez-Cambronero), by Veterans Affairs Merit Review grants, by a Medical Innovation Program from Wright State Univ. School of Medicine (to C. C. Paul and M. A. Baumann), and by the American Cancer Society, Ohio Division, and the Ohio State Board of Regents Research Challenge Program (to M. A. Baumann).

Address for reprint requests and other correspondence: J. Gómez-Cambronero, Dept. of Physiology and Biophysics, Wright State Univ. School of Medicine, 3640 Colonel Glenn Hwy., Dayton, OH 45435.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 22 March 2000; accepted in final form 15 August 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Cell Physiol 280(1):C183-C191