(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
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ABSTRACT
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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
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INTRODUCTION
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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 14, 5A, 5B, and 6) [2
3
4
].
STAT dimers recognize target-promoter elements related to the
-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
-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
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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 17 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-
(IFN-
; 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
, -ß, and JAK2
cDNAs and of an expressed sequence tag fragment of the human CIS gene.
The probes were labeled with (
-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
Denhardts 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
(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 manufacturers (Amersham)
instructions.
Electrophoretic mobility-shift assay (EMSA)
Human monocytes and 3- or 7-day differentiated macrophages were
stimulated with GM-CSF or IFN-
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 (
-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
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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-
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-
(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-
signaling. IFN-
induced very strong IRF-1 GAS
DNA-binding in monocytes and macrophages (Fig. 2
).
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.
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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
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 , 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 ) SDS-PAGE gels and transferred to
PVDF membranes. The membranes were stained with anti-GM-CSFR ,
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
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
|
|---|
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
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
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.
 |
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