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(Journal of Leukocyte Biology. 2002;71:511-519.)
© 2002 by Society for Leukocyte Biology

Granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced STAT5 activation and target-gene expression during human monocyte/macrophage differentiation

Anne Lehtonen, Sampsa Matikainen, Minja Miettinen and Ilkka Julkunen

Department of Microbiology, National Public Health Institute, Helsinki, Finland

Correspondence: Anne Lehtonen, Laboratory of Viral and Molecular Immunology, Department of Microbiology, National Public Health Institute, Mannerheimintie 166, FIN-00300 Helsinki, Finland. E-mail: Anne.Lehtonen{at}ktl.fi


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
GM-CSF signals through JAK2 and STAT5 and stimulates the expression of STAT5 target genes, such as pim-1 and CIS. Analyzed by EMSA, GM-CSF stimulation led to much stronger STAT5 DNA-binding to pim-1 or CIS GAS elements in primary human monocytes compared with mature macrophages. Similarly, GM-CSF-induced expression of pim-1 and CIS mRNAs was much stronger in monocytes. These differencies were not a result of downregulation of the GM-CSF receptor system or STAT5 expression, because monocytes and macrophages readily expressed GM-CSF receptor, JAK2, STAT5A, and STAT5B mRNAs and proteins. Monocytes expressed significant amounts of truncated STAT5 forms that took part in STAT5-DNA complex formation in GM-CSF-stimulated monocytes. This resulted in faster moving STAT5 complexes compared with macrophages in EMSA. Our results demonstrate that STAT5 isoform expression, GM-CSF-induced STAT5 activation, and STAT5 target-gene expression are altered significantly during monocyte/macrophage differentiation.

Key Words: signal transduction • cytokines • cellular differentiation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Monocytes and macrophages are important mediators of innate-immune responses because of their phagocytic potential and ability to produce various cytokines. They also take part in the induction of specific immune responses by presenting antigens to effector T cells [1 ]. Cytokines influence monocytes/macrophages in two different but partly overlapping ways: They regulate the proliferation and differentiation of these cells, and they are responsible for the functional activation of mature monocytes/macrophages. Cytokines exert their actions via specific receptors and signaling pathways. The Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway is one of the most commonly used (reviewed in refs. [2 3 4 ]). Upon ligand-binding, receptor-associated JAK tyrosine kinases are activated. Activated JAKs, in turn, phosphorylate latent cytoplasmic STAT proteins, which form homo- or heterodimers and translocate into the nucleus where they regulate the expression of cytokine-inducible genes. Presently, seven different STAT family members are known (STATs 1–4, 5A, 5B, and 6) [2 3 4 ]. STAT dimers recognize target-promoter elements related to the {gamma}-activated sequence (GAS) [5 ]. GAS-like elements have variable affinities for different STAT dimers.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage (M)-CSF regulate survival, proliferation, and differentiation of monocytes/macrophages. The GM-CSF receptor (GM-CSFR) is composed of a ligand-binding {alpha}-chain and the common ßc-chain of the cytokine receptor family [6 ]. GM-CSF activates JAK2 and STAT5 [7 , 8 ], but cell-type- and differentiation-specific differences have been described [9 , 10 ]. Two separate genes [8 ] encode for two STAT5 isoforms, STAT5A and STAT5B. In contrast to some other animal species [11 12 13 ] and some other STAT mRNAs [14 15 16 ], no STAT5 mRNA splice variants are known to exist in humans. However, full-length STAT5A and STAT5B proteins of 94 and 92 kDa in size are post-translationally modified by proteolytic processing [17 ] to smaller 77 kDa and 80 kDa forms, respectively [18 ]. These proteins have been shown to be expressed primarily in hematopoietic progenitor cells [17 , 18 ] and are suggested to act as transcriptional repressors [13 , 17 , 19 , 20 ]. GM-CSF/STAT5 pathway is known to regulate the expression of specific target genes such as pim-1 [21 , 22 ] and cytokine-inducible SH2-containing protein (CIS) [23 ].

In the present work, we have studied STAT5 expression and functional activity during differentiation of human primary monocytes to macrophages. We show that monocytes constitutively express the full-length and 77/80 kDa forms of STAT5 and that these proteins take part in the GM-CSF-induced GAS-binding complex formation in monocytes. We also demonstrate that GM-CSF-induced STAT5 DNA-binding and target-gene mRNA expression are stronger in monocytes than in macrophages, which express only the full-length STAT5 forms. Our results suggest that expression of 77/80 kDa STAT5 forms in monocytes does not necessarily lead to repression of STAT5 target-gene expression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture and cytokine stimulations
Leukocyte-rich buffy coats obtained from healthy blood donors were supplied by the Finnish Red Cross Blood Transfusion Service (Helsinki). Peripheral blood mononuclear cells were isolated by centrifugation on Ficoll-Hypaque gradient (Pharmacia Biotech, Uppsala, Sweden). For monocyte differentiation, mononuclear cells were allowed to adhere onto plastic six-well plates (20x106 cells/well; Falcon; Becton Dickinson, Franklin Lakes, NJ) for 1 h at 37°C in RPMI 1640 medium supplemented with penicillin (0.6 µg/ml), streptomycin (60 µg/ml), glutamine (2 mM), and HEPES (20 mM). After monocyte-binding, nonadherent cells were removed, and the wells were washed three times with cold phosphate-buffered saline (PBS), pH 7.4. Adherent cells were then grown for 1–7 days in Macrophage-SFM medium (Life Technologies, Gaithesburg, MD) supplemented with antibiotics. For macrophage differentiation, recombinant GM-CSF (Leucomax; Schering-Plough, Innishannon, Ireland) or recombinant M-CSF (R&D Systems, Abingdon, UK) was added to the medium at 10 ng/ml or 25 ng/ml, respectively. GM-CSF- or M-CSF-containing medium was removed from the cells 1 day before stimulation with GM-CSF. More than 90% of the cultured cells were monocyte/macrophages as determined by their morphology and CD14 expression [anti-CD14 fluorescein isothiocyanate monoclonal antibody (FITC mAb); Becton Dickinson; unpublished results]. Cells from individual blood donors were grown separately, but after stimulation experiments, they were pooled. All stimulations with GM-CSF (10 ng/ml) or interferon-{gamma} (IFN-{gamma}; 100 IU/ml) were done in RPMI 1640 medium for the times indicated in each experiment. To minimize interindividual variation among blood donors, all experiments were carried out using cells from four to six buffy coats.

RNA isolation and analysis
After GM-CSF treatment, the wells were washed with PBS, and the cells were lysed directly with guanidium isothiocyanate, followed by centrifugation through a CsCl cushion [24 , 25 ]. Total cellular RNA was recovered and quantified photometrically. Samples of total cellular RNA (10 µg) were size-fractionated on 1.0% denaturing formaldehyde-agarose gels and transferred to Hybond-N nylon membranes (Amersham, Buckinghamshire, UK). EtBr-staining of rRNA bands was used to ensure equal RNA loading. Probes used in Northern blot hybridizations were cDNA inserts of human STAT5A, STAT5B, Pim-1, and ß-actin genes and polymerase chain reaction (PCR) products of human GM-CSFR{alpha}, -ß, and JAK2 cDNAs and of an expressed sequence tag fragment of the human CIS gene. The probes were labeled with ({alpha}-32P)-dCTP (3000 Ci/mol; Amersham) using a random-primed DNA labeling kit (Boehringer-Mannheim, Mannheim, Germany). Hybridizations were performed in conditions of high stringency [50% formamide, 5x Denhardt’s solution, 5x SSPE, and 0.5% sodium dodecyl sulfate (SDS) at 42°C]. Filters were washed twice with 1x standard saline citrate (SSC) - 0.1% SDS at room temperature for 0.5 h and once at 60°C for 0.5 h. Kodak X-OMAT AR film was used for autoradiography at -70°C with intensifying screens.

Immunoprecipitation, gel electrophoresis, and Western blotting
Monocytes and macrophages were stimulated with GM-CSF for 15 min or left untreated, collected with a cell scraper in cold PBS, and lysed in immunoprecipitation buffer [50 mM Tris, pH 7.4, containing 150 mM NaCl, 5 mM ethylenediaminetetraacetate, and 1% Triton X-100] on ice for 15 min. Cell lysates were cleared by centrifugation and immunoprecipitated with polyclonal rabbit anti-human STAT5 antibody (sc-835X recognizing the C-terminal end of STAT5A and STAT5B; Santa Cruz Biotechnology, Santa Cruz, CA) with STAT5 isoform-specific anti-STAT5A or anti-STAT5B (71-2400 and 71-2500; ZYMED, San Francisco, CA) antibodies or with antibodies against the phosphotyrosyl form of JAK2 (#07-123; Upstate Biotechnology, Lake Placid, NY).

SDS-polyacrylamide gel electrophoresis (PAGE) was carried out using the Laemmli buffer system [26 ] on 8% or 10% polyacrylamide gels. Proteins separated on gels were transferred onto Immobilon-P [polyvinylidene difluoride (PVDF)] membranes (Millipore, Bedford, MA) with an Isophor electrotransfer apparatus (Hoefer Scientific Instruments, San Francisco, CA) at 200 mA for 2 h. For detection of tyrosine phosphorylation, primary antiphosphotyrosine antibody (sc-7020, 1/200 dilution; Santa Cruz Biotechnology) was allowed to bind in PBS containing 3% bovine serum albumin for 1 h at room temperature (RT) followed by secondary antibody-binding with biotin-SP-conjugated goat anti-mouse immunoglobulin G (IgG; 115-066-072, 1/10 000; Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 h at RT. After this, peroxidase-conjugated streptavidin (016-030-084, 1/1000; Jackson) was allowed to bind for 1 h at RT. Other primary antibodies were anti-GM-CSF-R{alpha} (mAb 1006, 2 µg/ml; Chemicon, Temecula, CA), anti-GM-CSF-Rß (1:500; sc-676; Santa Cruz Biotechnology), and anti-JAK2 (1/2000; a kind gift from Dr. O. Silvennoinen). Horseradish peroxidase-conjugated anti-mouse (62-6620, 1/1000; ZYMED) and anti-rabbit (P0448, 1/2000; Dako A/S, Glostrup, Denmark) Igs were used as secondary antibodies. The bands were visualized on Amersham Hyper-Max film using the enhanced chemiluminescence system, according to the manufacturer’s (Amersham) instructions.

Electrophoretic mobility-shift assay (EMSA)
Human monocytes and 3- or 7-day differentiated macrophages were stimulated with GM-CSF or IFN-{gamma} for different periods of time, and nuclear extracts were prepared as previously described [27 ]. Nuclear protein/DNA-binding reactions were performed as described previously [28 ] in a 20 µl vol containing 5 µg nuclear extract protein. Human IRF-1 GAS (5'-AGCTTCAGCCTGATTTCCCCGAAATGACGGA-3'), pim-1 GAS (5'-ACACACATCCCTTCCCAGAAATCAGGATTC-3'), CIS GAS1 (5'-CCCCGTTTTCCTGGAAAGTTTTGGAAATCTGT-3'), and CIS GAS2 (5'-CCGCGGTTCTAGGAAGACGCTGCTTCCGGGAAGGGCTGG-3') oligonucleotide probes were from DNA Technology ApS (Aarhus, Denmark). The putative human CIS GAS element sequences were found by database searches. We have named the distal element CIS GAS-1 and the proximal element CIS GAS-2. The mouse [23 ] and human GAS sequences were found to be identical, although there were minor differences in the flanking nucleotides. The probes were end-labeled with ({gamma}-32P)-dATP (3000 Ci/mol; Amersham) by T4 polynucleotide kinase. The binding reaction was done at RT for 0.5 h. Complexes were separated from the free probe in 6% nondenaturing, low ionic-strength PAGE gels in 0.25x TBE buffer. Gels were dried, and bands were visualized by autoradiography. Unless otherwise indicated, the extracts from cells stimulated for 0.5 h were used for supershift assays. For this, nuclear protein extracts were incubated with antibodies for 1 h on ice prior to the addition of the radiolabeled probe. Anti-STAT antibodies used in supershift assays were anti-STAT1 (sc-346X), anti-STAT3 (sc-482X), and anti-STAT5 (sc-835X). For competition EMSA analysis, 1-, 3-, 10-, 30-, or 100-fold molar excess of competing oligonucleotide (CIS GAS-1) was added to the binding reactions.

DNA affinity purification of STAT5
Both strands of CIS GAS-1 element were synthesized with BamHI overhangs, and the lower strand oligonucleotide was 5'-biotinylated. Oligonucleotides were annealed in 0.5 M NaCl. The annealed probe was incubated with streptavidin-agarose beads (Neutravidin; Pierce, Rockford, IL) at +4°C for 2 h in a ratio to yield maximum saturation of the beads with the biotinylated oligonucleotide. Monocytes and macrophages were treated with GM-CSF for 15 min of left untreated. Cells were collected and treated as described by Rosen et al. [29 ]. Cell extracts were incubated with the agarose beads saturated with the oligonucleotide for 2 h at +4°C. After washing, the samples were released by SDS sample buffer and run on 10% SDS-PAGE gels, followed by detection with polyclonal rabbit anti-human STAT5 antibody (sc-835X; Santa Cruz Biotechnology).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
GM-CSF-induced STAT DNA-binding is altered during monocyte/macrophage differentiation
We stimulated 1-day-old monocytes and GM-CSF-differentiated, 3- or 7-day-old macrophages with GM-CSF for 0.5 h. Nuclear extracts were prepared and analyzed by EMSA with GAS elements from STAT5-responsive target genes, pim-1 (pim-1 GAS; Fig. 1 A ) and CIS (CIS GAS-1 and CIS GAS-2; Fig. 1B and 1C ). GM-CSF induced a very strong GAS DNA-binding complex in 1-day monocyte-cell extracts with all elements, but only the CIS GAS-1 element was able to react with macrophage extracts to produce a detectable DNA-binding complex (Fig. 1B) . The mobility of the GM-CSF-induced complex seen in 1-day monocytes was also faster compared with partially (3-day) or fully (7-day) differentiated macrophages.



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  		Figure 1. EMSA analysis of GM-CSF-induced GAS DNA-binding during monocyte-to-macrophage differentiation. Monocytes and 3- and 7-day GM-CSF-differentiated macrophages were stimulated with GM-CSF for 0.5 h or left untreated, and nuclear extracts were analyzed using pim-1 GAS (A), CIS GAS-1 (B), and CIS GAS-2 (C) probes in EMSA.

 
Hyporesponsiveness to cytokine stimulation is not a general feature of in vitro GM-CSF-differentiated macrophages
Because macrophages responded to GM-CSF with less intensity than monocytes, we wanted to ascertain that the macrophage population was not generally nonresponsive to cytokine stimulation. IFN-{gamma} is the major activator of monocyte/macrophages and, hence, a functionally important cytokine. Therefore, we stimulated monocytes and GM-CSF-differentiated, 7-day macrophages with IFN-{gamma} (100 IU/ml) for times indicated in the figure. Nuclear extracts were analyzed by EMSA with an IRF-1 GAS element, a well-studied target element of IFN-{gamma} signaling. IFN-{gamma} induced very strong IRF-1 GAS DNA-binding in monocytes and macrophages (Fig. 2 ).



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  		Figure 2. Monocytes and macrophages respond in a similar manner to IFN-{gamma} stimulation. Monocytes and 7-day GM-CSF-differentiated macrophages were stimulated with IFN-{gamma} for times indicated in the figure or left untreated, and nuclear extracts were analyzed by EMSA with the IRF-1 GAS element. 0I and 0II are untreated control cells collected at 0.5 and 24 h, respectively.

 
Kinetics of GM-CSF-induced STAT5 target-gene expression in monocytes and macrophages
The EMSA results obtained with STAT5 target-DNA elements prompted us to study mRNA expression of pim-1 and CIS by Northern blotting in GM-CSF-stimulated monocytes and macrophages. The basal expression levels of pim-1 and CIS mRNAs were very low in monocytes and macrophages (Fig. 3 ). In monocytes, GM-CSF stimulation resulted in rapid and abundant expression of pim-1 and CIS mRNAs within 1 h. Pim-1 expression peaked at 2 h after stimulation and declined thereafter, whereas CIS mRNA levels remained elevated for 24 h. In macrophages, GM-CSF stimulation did not induce expression of pim-1 mRNA. A weak induction of CIS mRNA was detected in macrophages, in accordance with the EMSA data showing STAT5-binding to the CIS GAS-1 element (Fig. 1) .



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  		Figure 3. Expression kinetics of pim-1 and CIS mRNAs in human primary monocytes and macrophages. One-day monocytes and 7-day GM-CSF-differentiated macrophages were stimulated with GM-CSF for times indicated in the figure, and total cellular RNA was isolated. For Northern blot analyses, 10 µg samples of total cellular RNA were separated on 1% agarose-formaldehyde gels, transferred onto nylon membranes, and hybridized with pim-1, CIS, and ß-actin probes. 0I, 0II, and 0III represent unstimulated control cells collected at 1, 4, and 24 h, respectively.

 
M-CSF-differentiated macrophages display decreased sensitivity to GM-CSF stimulation
Because GM-CSF stimulation led to weaker GAS DNA-binding response and target-gene mRNA expression in macrophages compared with monocytes, we considered the possibility that GM-CSF-induced differentiation would desensitize macrophages to restimulation with this cytokine. M-CSF uses a different receptor system than GM-CSF [30 ] and is also able to induce differentiation of monocytes to macrophages. Again, in monocytes, strong STAT5 DNA-binding to pim-1 GAS and CIS GAS-1 and -2 was seen. In M-CSF-differentiated, GM-CSF-stimulated macrophages, only the CIS GAS-1 element bound STAT5 (Fig. 4 A ), similar to the DNA-binding pattern of GM-CSF-differentiated cells. GM-CSF-induced pim-1 and CIS mRNA levels were also clearly lower in M-CSF-differentiated (as well as in GM-CSF-differentiated) macrophages than in monocytes (Fig. 4B ; compare with Fig. 3 ).



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  		Figure 4. STAT5 DNA-binding and target-gene mRNA expression in M-CSF-differentiated macrophages. (A) Monocytes and 3- and 7-day M-CSF-differentiated macrophages were stimulated with GM-CSF for 0.5 h or left untreated, and nuclear extracts were analyzed using pim-1 GAS, CIS GAS-1, and CIS GAS-2 probes in EMSA. (B) One-day monocytes and 7-day M-CSF-differentiated macrophages were stimulated with GM-CSF for times indicated in the figure, and total cellular RNA was isolated. For Northern blot analyses, 10 µg samples of total cellular RNA were separated on 1% agarose-formaldehyde gels, transferred onto nylon membranes, and hybridized with pim-1 and CIS probes. 0I and 0II represent unstimulated control cells collected at 1 and 24 h, respectively.

 
Kinetics of GAS DNA-binding activity and identification of GAS DNA-binding protein composition in GM-CSF-stimulated monocytes and macrophages
Because the monocyte/macrophage differentiation process clearly affected the intensity of GM-CSF-induced STAT DNA-binding and mRNA expression of STAT5-regulated genes, pim-1 and CIS, we studied the kinetics of STAT complex formation in EMSA in more detail and identified the composition of the complex by supershift EMSA analysis. In monocytes, GM-CSF-inducible DNA-binding complexes binding to pim-1 GAS, CIS GAS-1, and CIS GAS-2 elements appeared at 0.5 h after stimulation and persisted for 24 h (Fig. 5A 5B 5C ). In macrophages, with CIS GAS-1, the intensity of the GM-CSF-induced complex was weaker compared with monocytes, but it was still detectable after 24 h of stimulation (Fig. 5B) . With pim-1 GAS and CIS GAS-2, no GM-CSF-inducible complexes in macrophages were observed. Consistent with results shown in Figure 1 , monocyte STAT complexes had faster mobility compared with macrophages.



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  		Figure 5. Kinetics of GM-CSF-induced DNA-binding to STAT5 target-gene promoter GAS elements. Monocytes and 7-day GM-CSF-differentiated macrophages were stimulated with GM-CSF for times indicated in the figure or left untreated, and nuclear extracts were analyzed by EMSA with pim-1 GAS (A), CIS GAS-1 (B), and CIS GAS-2 (C) elements. 0I and 0II are untreated control cells collected at 0.5 and 24 h, respectively. (D) Nuclear extracts from cells stimulated with GM-CSF for 0.5 h were supershifted with anti-STAT1, -STAT3, and -STAT5 antibodies using the CIS GAS-1 element.

 
To identify STAT protein composition of GM-CSF-inducible GAS-binding complexes, we performed supershift EMSA analyses (Fig. 5D) . In monocytes, the antibody recognizing the C-terminus of STAT5 showed a partial supershift of the DNA-binding complex. In macrophages, instead, complete supershift was seen with the same antibody. Anti-STAT1 or anti-STAT3 antibodies showed no supershift activity, suggesting that in monocytes/macrophages, GM-CSF could only activate STAT5 proteins.

We also performed EMSA analysis with the CIS GAS-1 oligonucleotide using 1- to 100-fold molar excess of the same oligonucleotide as a competitor in the binding reaction (Fig. 6 ). In monocytes, 100-fold molar excess of competing oligonucleotide was needed to completely block the formation of the DNA-binding complex, whereas in macrophages, tenfold molar excess efficiently competed for complex-binding. This shows that the amount of the DNA-binding complex in monocytes is clearly higher than in macrophages.



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  		Figure 6. Competition EMSA analysis. Nuclear extracts were prepared from GM-CSF-stimulated (0.5 h) and control monocytes and 7-day macrophages and were analyzed by EMSA with CIS GAS-1 oligonucleotide. Unlabeled CIS GAS-1 oligonucleotide was added to the binding reaction in 1- to 100-fold molar excess as indicated in the figure.

 
Expression of GM-CSFR components and activation of JAK2 during monocyte/macrophage differentiation
Because GM-CSF-induced activation of STAT DNA-binding was significantly reduced during monocyte/macrophage differentiation, we wanted to see if expression of GM-CSF-receptor components or expression or activation of JAK2 was altered during differentiation. Adherent monocytes were cultured in the presence or absence of GM-CSF, and total cellular RNA was isolated. No significant changes in the expression levels of GM-CSFR{alpha} and GM-CSFRß mRNAs were observed between monocytes and differentiated macrophages when mRNA levels were compared with ß-actin mRNA levels (Fig. 7 A ). JAK2 mRNA was expressed at a low basal level that also remained unchanged (Fig. 7A) .



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  		Figure 7. Expression of GM-CSFR chains and expression and activation of JAK2 in monocytes and macrophages. (A) Total cellular RNA was isolated from monocytes and GM-CSF-differentiated, 3- and 7-day-old macrophages. For Northern blot analyses, 10 µg samples of total cellular RNA were separated on 1% gels, transferred onto nylon membranes, and subsequently hybridized with GM-CSFR{alpha}, GM-CSFRß, JAK2, and ß-actin probes. (B) For Western blot analyses, protein samples from monocytes and macrophages were run on 8% (for GM-CSFRß and JAK2) or 10% (for GM-CSFR{alpha}) SDS-PAGE gels and transferred to PVDF membranes. The membranes were stained with anti-GM-CSFR{alpha}, anti-GM-CSFRß, and anti-JAK2 antibodies. (C) Lysates from GM-CSF-stimulated and control cells from monocytes and macrophages were immunoprecipitated with anti-P-Y JAK2 antibody. The precipitates were run on 8% SDS-PAGE and transferred to a PVDF membrane that was stained with anti-JAK2 antibody.

 
We also prepared protein samples from monocytes and macrophages and analyzed them by Western blotting. Protein expression of GM-CSFR{alpha} and GM-CSFRß receptor chains was higher in macrophages than in monocytes (Fig. 7B) . Because JAK2 protein levels were somewhat lower in macrophages than in monocytes (Fig. 7B) , we studied GM-CSF-induced activation of JAK2 by direct immunoprecipitation of the phosphorylated active form of JAK2 (Fig. 7C) . In monocytes, tyrosine phosphorylation of JAK2 was at an undetectable level, but in macrophages, it was readily detectable.

Expression of STAT5A and STAT5B mRNAs and proteins in monocytes and macrophages
To study whether the differences in STAT activation in monocytes and macrophages seen in EMSA were a result of differential expression of STAT5 isoforms, we analyzed STAT5A and STAT5B mRNA and protein expression during differentiation. Total cellular RNA from monocytes and macrophages was isolated and analyzed by Northern blotting. No significant changes in the expression levels of STAT5A or STAT5B mRNAs were observed during monocyte differentiation to macrophages (Fig. 8 A ). Splice variants of STAT5A or STAT5B mRNAs were not found.



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  		Figure 8. Expression of STAT5A and STAT5B during differentiation of monocytes to macrophages. (A) Total cellular RNA was isolated from 1- and 3-day-old monocytes and GM-CSF-differentiated, 1- to 7-day-old macrophages. For Northern blot analyses, 10 µg samples of total cellular RNA were separated on 1% gels, transferred onto nylon membranes, and subsequently hybridized with STAT5A, STAT5B, and ß-actin probes. (B) Lysates from unstimulated monocytes and 7-day macrophages were immunoprecipitated with three different antibodies, anti-C-STAT5 (recognizing the C-terminus of STAT5A and STAT5B) and isoform-specific anti-STAT5A and anti-STAT5B antibodies. Immunoprecipitated proteins were separated on 10% SDS-PAGE gels and transferred onto PVDF membranes. The blots were subsequently stained with antibodies indicated in the figure.

 
To study STAT5 protein expression, we immunoprecipitated STAT5 from monocytes and mature macrophages with three different antibodies (see Materials and Methods). After SDS-PAGE and Western blotting, the membranes were stained with antibodies used for immunoprecipitation. Monocytes and macrophages expressed the expected full-length 94/92 kDa STAT5 protein forms. Additionally, in monocytes, smaller STAT5 forms of approximately 77/80 kDa were found (Fig. 8B) . In monocytes, the intensity of 94 kDa and 77 kDa STAT5A bands was equal, whereas the full-length form predominated for STAT5B. The presence of the 77/80 kDa STAT5 forms was not dependent on activation of the proteins by tyrosine phosphorylation, because the cells were not stimulated with GM-CSF. No major differences in the overall expression levels of full-length STAT5 forms were detected between monocytes and macrophages.

GM-CSF stimulation induces tyrosine phosphorylation and homo- and heterodimerization of STAT5A and STAT5B
Cell lysates from monocytes and 7-day-old macrophages, treated with GM-CSF for 15 min or left untreated, were immunoprecipitated with anti-STAT5 isoform-specific antibodies followed by Western blotting with an antiphosphotyrosine antibody. In monocytes, only the full-length forms of STAT5A and STAT5B were found to be tyrosine-phosphorylated in response to GM-CSF stimulation (Fig. 9 A ), despite the expression of the shorter STAT5 forms. GM-CSF-induced tyrosine phosphorylation of STAT5B was clearly more pronounced in monocytes than in macrophages. STAT5A immunoprecipitates were also cross-stained with anti-STAT5B antibodies and vice versa. STAT5A- or STAT5B-specific antibodies were able to immunoprecipitate both STAT5 isoforms, indicating that heterodimerization between STAT5A and STAT5B had taken place (Fig. 9B) .



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  		Figure 9. GM-CSF-induced tyrosine phosphorylation, heterodimerization, and DNA-binding of STAT5 isoforms in monocytes and macrophages. Monocytes and 7-day macrophages were treated with GM-CSF for 15 min or left untreated. (A) Cell lysates were prepared and immunoprecipitated with STAT5A- and STAT5B-specific antibodies. Immunoprecipitated proteins were run on 10% SDS-PAGE and transferred onto PVDF membranes. The blots were subsequently stained with antiphosphotyrosine antibodies and STAT5 isoform-specific antibodies. Arrows indicate 94-kDa STAT5A and 92-kDa STAT5B proteins. (B) STAT5A immunoprecipitates were stained with anti-STAT5B antibodies and vice versa. (C) Cell lysates were incubated with agarose-immobilized CIS GAS-1 oligonucleotide. Bound proteins were run on 10% SDS-PAGE and transferred to PVDF membranes that were subsequently stained with C-terminus-specific anti-STAT5 antibody.

 
The faster mobility of the GM-CSF-induced STAT5 complex in monocytes compared with macrophages could be explained by participation of the 77/80-kDa forms in the DNA-binding complex in monocytes. Although we could not detect tyrosine phosphorylation of the 77/80 kDa forms of STAT5 directly, we precipitated proteins from GM-CSF-stimulated monocytes and macrophages based on their affinity to the CIS GAS-1 DNA element (Fig. 9C) . Western blotting with C-terminus-specific STAT5 antibody revealed that in monocytes, the 77/80-kDa STAT5 forms took part in the DNA-binding complex, in addition to the full-length forms.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we demonstrate that GM-CSF-induced STAT5 activation, STAT5-isoform expression, and STAT5 target-gene activation change during the in vitro human monocyte/macrophage differentiation. We observed that the mobility of STAT5 DNA-binding complexes was slower in macrophages than in monocytes as a result of participation of the smaller 77/80-kDa STAT5 forms in the DNA-binding complexes in monocytes. We also found that GM-CSF-induced STAT5 DNA-binding activity was reduced significantly in 7-day differentiated macrophages compared with monocytes. The situation was similar in M-CSF-differentiated cells, implying that these phenomena are likely to be differentiation-related and do not result from prolonged GM-CSF treatment. Expression of GM-CSFR-chain mRNAs was not altered significantly during differentiation. Rather, GM-CSFR protein expression was higher, and GM-CSF-induced JAK2 activation was more pronounced in macrophages compared with monocytes, further suggesting that a functional GM-CSFR system existed in macrophages.

In our cellular system, 77/80-kDa forms of STAT5 were expressed constitutively in monocytes but not in macrophages. Immature hematopoietic progenitor cells express low molecular-weight STAT5 isoforms most prominently [18 , 29 , 31 ], and this has been associated with inhibition of target-gene expression [13 , 17 , 19 , 20 ]. Our data obtained in normal human monocytes do not support this concept, because despite expression of truncated STAT5 forms, GM-CSF stimulation of monocytes resulted in strong activation of STAT5 DNA-binding and induction of mRNA expression of two STAT5 target genes, pim-1 and CIS. In macrophages, GM-CSF-induced STAT5 DNA-binding and target-gene expression (Figs. 1 3 and 5) were weak, although the cells expressed only the full-length STAT5 isoforms.

A constitutively active nuclear protease taking part in STAT5 proteolytic processing has been described [32 ], although the smaller STAT5 forms have been suggested to be experimental artefacts, at least in mammary gland tissue [33 ]. We found that monocytes constitutively expressed full-length and truncated forms of STAT5A and STAT5B proteins, but apparently only the full-length forms (94/92 kDa) were phosphorylated in response to GM-CSF treatment. Previous studies have suggested that GM-CSF activates full-length and truncated forms of the STAT5A protein [29 ]. Smaller STAT5 isoforms have also been shown to have DNA-binding capacity [18 , 29 ]. When we precipitated proteins based on their affinity to the CIS GAS-1 DNA element, we found that the 77/80-kDa STAT5 forms also bound to the DNA, indirectly demonstrating their activation by GM-CSF in monocytes and their potential role in enhancing target-gene expression. We also observed that STAT5A and STAT5B proteins were capable of forming heterodimers (Fig. 9B) , although homodimerization has been suggested to be the dominant form of association following GM-CSF stimulation [29 , 34 ].

GM-CSF-induced STAT5B activation was clearly stronger in monocytes compared with macrophages. The cytoplasmic regions of GM-CSFR{alpha} and ßc have been shown to affect the composition of STAT5 complexes to be activated [35 ]. Subtle modifications in the cytoplasmic domains could be responsible for decreased affinity for STAT5 in general and STAT5B in particular in macrophages. It may be that activated STAT5B may be required for GM-CSF-induced pim-1 and CIS gene activation. In the context of the CIS promoter, it has been shown that STAT5B binds to CIS GAS-1 and GAS-2 as dimers, whereas STAT5A binds to CIS GAS-2 preferentially as tetramers [36 ]. In this study, we observed that in monocytes, the distal CIS GAS-1 element bound STAT5 more strongly than the proximal CIS GAS-2 in response to GM-CSF stimulation. This could reflect the preference of STAT5B to be activated over STAT5A. Additionally, STAT5 proteins are known to interact with transcriptional cofactors or basal transcription machinery proteins [37 ]. In some cases, this interaction might require that STAT proteins are present as dimers, as STAT5B on the CIS promoter, and STAT tetramerization could block this interaction.

CIS protein is a member of the CIS/suppressor of cytokine signaling family [38 , 39 ], and its expression is under STAT5 regulation [23 ]. The role of CIS protein in normal cell functions still remains unresolved. However, there are indications that CIS has a role in tumor suppression and in cell-growth inhibition [40 , 41 ]. CIS creates a negative feedback loop by masking STAT5 docking sites on the receptor and interfering with STAT5 activation by erythropoietin (Epo) and interleukin-3 [40 ]. We did not see inhibition of CIS mRNA expression in monocytes during a period of 24 h, so it is possible that the CIS protein may not block GM-CSF-induced STAT5 activation as efficiently. Therefore, this could be a result of lower affinity of the CIS protein for GM-CSFR than for EpoR.

Bone marrow-derived cells of mice with disrupted STAT5A or STAT5A/STAT5B genes display decreased proliferative responses to GM-CSF [42 , 43 ], indicating an important role for STAT5 in GM-CSF signaling, although STAT5 is apparently not involved in monocyte/macrophage differentiation [43 ]. GM-CSF induces expression of Pim-1, a serine/threonine kinase known to enhance proliferation of myeloid cells [21 , 44 ]. Previously, constitutive DNA-binding of a mutant STAT5A protein [45 ] was shown to be associated with growth factor-independent cell proliferation, and this effect was mediated by STAT5-induced Pim-1 expression. Therefore, it is likely that Pim-1 is involved in mediating proliferative responses in monocytes. Accordingly, GM-CSF induced STAT5 DNA-binding to pim-1 GAS and activated pim-1 mRNA expression in monocytes but failed to do so in macrophages, suggesting that in mature macrophages, the expression of proliferation-associated genes, such as pim-1, is no longer needed. In monocytes, the activation of these genes seemed to require strong, initial STAT5 activation. Hence, in macrophages, one way to inhibit expression of these genes would be to reduce activation of STAT5. A lower STAT5 activation level may be sufficient to induce expression of GM-CSF-regulated cell-survival genes in macrophages.

STAT5, which is activated by a number of different ligands, has been recognized as a factor closely linked to cellular proliferation and differentiation [18 , 29 , 46 47 48 49 ]. STAT5 is regulated at multiple levels: existence of two isofoms, STAT5A and STAT5B, with distinct DNA-binding specificities [49 ], expression of low molecular-weight forms of both isoforms [18 ], and the ability to form homo- and heterodimeric as well as tetrameric complexes [36 , 50 ]. The versatility of target-element sequences in natural promoters and other interacting transcription factors adds to this complexity. In this work, we have shown that by altering the overall activation level of STAT5, cells can regulate gene expression further by GM-CSF/STAT5. All of these factors contribute to fine-tuning target-gene expression in different cell types and during normal cellular differentiation, of which this work gives an example.


   ACKNOWLEDGEMENTS
 
This study was supported by the Medical Research Council of the Academy of Finland, the Finnish Cultural Foundation, the Emil Aaltonen Foundation, and the Finnish Cancer Society. We thank Drs. A. Meinke and T. Decker (Vienna, Austria) for providing STAT5A and STAT5B cDNAs and Dr. A. Miyajima (Tokyo, Japan) for the GM-CSFR{alpha} and -ß cDNAs. We also thank Dr. Tapani Hovi for helpful discussions and Ms. Katja Moilanen, Ms. Valma Mäkinen, Ms. Teija Westerlund, and Ms. Marika Yliselä for excellent technical assistance.

Received February 27, 2001; revised October 28, 2001; accepted October 30, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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