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B p651
*
Division of Hematopoietic Factors and
Department of Hematology/Oncology, Institute of Medical Science, University of Tokyo, Tokyo, Japan;
Department of Host Defense, Core Research for Engineering, Science, and Technology of Japan Science and Technology Corporation, Research Institute for Microbial Diseases, Osaka University, Suita, Japan; and
Division of Hematology, Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B
p65 (RelA) to the promoter region of IL-6. These findings indicate that
STAT5A cooperates with Rel/NF-B to induce production of IL-6,
thereby inducing macrophage differentiation of M1 cells in an autocrine
manner. In summary, we have shown a novel mechanism by which STAT5
induces its pleiotropic functions. Cytokines |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A myeloblastic leukemia cell line, M1, was generated from a spontaneous
leukemia that arose within the SL strain of mice (11) and
can be induced to undergo terminal differentiation into macrophages,
which is associated with growth arrest and apoptosis of mature cells
(12) by IL-6, LIF, or human oncostatin M (OSM)
(13, 14, 15). Ectopic expression of the 
and 
subunits
of the human GM-CSF receptor in M1 cells confers a potential to
terminally differentiate into macrophages upon stimulation with human
GM-CSF (16). Because STAT5 is one of the key molecules
that are activated in response to GM-CSF (6, 17), we asked
whether STAT5 is involved in monocytic differentiation of M1 cells.
STAT5A1*6 induced macrophage differentiation of M1 cells, but through
an unexpected mechanism. Activation of STAT5 leads to IL-6 production
following enhancement of the DNA binding activity of NF-B p65,
suggesting a potential role of STAT5A to regulate
IL-6-induced hemopoietic cell differentiation within the
myelomonocytic lineage.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The murine myeloid leukemia cell line M1 was grown in DMEM (Life Technologies, Rockville, MD) containing 10% FCS. Mouse (m)IL-3-dependent Ba/F3 cells were maintained in RPMI 1640 medium containing 10% FCS and 1 ng/ml rmIL-3 produced in silk worm (18). An ecotropic retrovirus packaging cell line BOSC23 (19) was maintained in DMEM containing 10% FCS and guanine phosphoribosyltransferase selection reagents (Specialty Media, Lavallette, NJ). The cells were transferred into DMEM containing 10% FCS, without guanine phosphoribosyltransferase selection reagents, 2 days before transfection. The mIL-6, goat anti-mIL-6 neutralizing Ab (anti-IL-6 Ab), and goat anti-mLIF neutralizing Ab (anti-LIF Ab) were obtained from R&D Systems (Minneapolis, MN). PE-conjugated rat anti-mouse CD14 Ab were purchased from BD PharMingen (San Diego, CA).
Retrovirus vectors
A bicistronic retrovirus vector pMX-IRES-EGFP was constructed to transduce a gene together with an enhanced green fluorescent protein (EGFP) as previously described (10). Complementary DNAs for the wild-type mSTAT5A and STAT5A1*6 were inserted into EcoRI and NotI sites of the pMX-IRES-EGFP to construct pMX-STAT5A-IRES-EGFP and pMXSTAT5A1*6-IRES-EGFP.
Production of retroviruses and infection with these viruses
High-titer retroviruses containing the wild-type STAT5A and STAT5A1*6 were produced in a transient retrovirus packaging cell line BOSC23 as previously described (20). For infection, M1 cells (1 x 106) were incubated for 6 h with 10 ml of the retroviruses harboring the wild-type and STAT5A1*6 in the presence of 10 µg/ml hexadimethrine bromide (Sigma, St. Louis, MO). Ten milliliters of fresh growth medium was added to the culture, and incubation was continued for 18 h. The cells were washed, refed with growth medium, and allowed to grow for 1 more day before cell sorting.
Cell sorting and flow cytometry
Sorting of EGFP-positive cells was done as previously described (21). Briefly, 2 days after virus infection, cells were washed twice with PBS and suspended in PBS containing 1% BSA. Retrovirally transduced cells were sorted based on GFP expression on a FACSVantage (BD Biosciences, Mountain View, CA). The sorted cells (1 x 104) were washed twice with PBS, resuspend in growth medium, and cultured for 2 more days (4 days after virus infection). A half of the sorted population was used to confirm GFP expression using FACSCalibur (BD Biosciences), and the other half was expanded and used for further analysis.
Immunoprecipitation and Western blotting
Immunoprecipitation, gel electrophoresis, and immunoblotting were done as previously described (10) with minor modifications. Exponentially growing cells were washed in PBS so as to be free of serum and growth factors, lysed in a buffer (5 x 106 cells/ml), and incubated on ice for 30 min. Cell lysates were clarified by centrifugation for 15 min at 12,000 x g before incubation at 4°C overnight with the anti-STAT5A polyclonal Ab (R&D Systems) or the control rabbit whole IgG, and protein A-Sepharose. The immunoprecipitates were washed three times with a lysis buffer, subjected to SDS-PAGE, and electrophoretically transferred onto Immobilon filters (Millipore, Bedford, MA). After blocking in a solution containing 3% BSA, the filter was probed with an anti-phosphotyrosine Ab 4G10 (Upstate Biotechnology, Lake Placid, NY), stripped, and reprobed with the anti-STAT5A Ab to verify the amount loaded. The filter-bound Ab was detected using the ECL system (Amersham, Arlington Heights, IL).
EMSA in M1 cells
The cells were lysed in a binding buffer (2 x 107 cells/ml) containing 0.5% Nonidet P-40, 50 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 150 mM NaCl, 100 µM Na3VO4, 50 mM NaF, 1 mM DTT, 0.4 mM PMSF, 3 µg/ml aprotinin, 2 µg/ml pepstatin A, 1 µg/ml leupeptin, and 10% glycerol and were centrifuged at 12,000 x g for 30 min to remove insoluble materials. The cell extracts (4 µl/each reaction) were incubated with 1 ng of Klenow-labeled DNA carrying the following STAT5 optimal binding sequence: 5'-GATCCGAATTCCAGGAATTCA-3' and 3'-GCTTAAGGTCCTTAAGTCTAG-5'. For supershift analysis, cell extract in the binding buffer was incubated with the anti-STAT5A rabbit anti-serum (R&D Systems) or normal rabbit serum, as a control, for 30 min on ice before addition of poly(dI-dC). Samples were separated on a 4.5% polyacrylamide gel in 2.2x TBE (1x TBE = 50 mM Tris-borate and 1 mM EDTA) and autoradiographed.
Luciferase reporter assay
Ba/F3 cells were transiently transfected by electroporation at
960 µF and 300 V with 10 µg of a reporter plasmid carrying a
luciferase gene driven by the IL-6 promoter, 3 µg of a
-galactosidase reporter plasmid with the Rous sarcoma virus
long-terminal repeat promoter, and 10 µg of test DNAs at room
temperature in RPMI 1640 supplemented with 10 µg/ml DEAE-dextran.
After a 12-h recovery period in the IL-3-containing medium, cells were
incubated in RPMI 1640 supplemented with 0.5% BSA for 12 h, or
stimulated with 2 ng/ml mIL-3 for the last 6 h before cell lysates
were prepared. Cell lysates were then subjected to luciferase (Promega,
Madison, WI) and -galactosidase (Stratagene, La Jolla, CA) assays.
Transfection efficiency was normalized with the -galactosidase
activity. The wild-type IL-6 promoter (k0) and its deletion mutants
(k4, k18, and k9) were as previously described (22).
Site-directed mutagenesis of k0 and k9 was done using QuikChange
(Stratagene) and the oligonucleotide primers
5'-ATCAAATGTAATATTTTCCCATGAGTCTGA-3' and
5'-TGAGACTCATGGGAAAATATTACATTTGAT-3'.
Transfection and preparation of nuclear extracts in COS cells
Cells were cotransfected with 1 µg of human Epo receptor gene,
1 µg pEF-NF-B p65, and 1 µg of either pMX, pMX/WT-STAT5A, or
pMX/STAT5A1*6 using FuGENE 6 Transfection Reagent (Roche Diagnostics,
Indianapolis, IN). Forty-eight hours after transfection, cells were
stimulated with recombinant human Epo (20 U/ml) for 30 min or left
untreated before harvesting. Nuclear extracts were then prepared as
previously described (10).
EMSA in COS, M1, and MD cells
Double-stranded oligonucleotides corresponding to the NF-B
site of the IL-6 promoter were used as DNA probes. EMSA was done using
10 µg of nuclear extracts in 25 µl of a reaction mixture containing
10 mM HEPES (pH7.8), 4% glycerol, 0.02 mM EDTA, 84 mM KCl, 1 mM
MgCl2, 3 µg of poly(dI-dC), 5 mM DTT, and 10
fmol of radioactive probe. The mixture was incubated on ice for 20 min
and placed onto a 4.5% polyacrylamide gel (0.25x TAE). For supershift
experiments, 1 µl of anti-NF-B p65 Ab, anti-c-Rel Ab
(Santa Cruz Biotechnology, Santa Cruz, CA), control rabbit polyclonal
IgG, or anti-STAT5A Ab (R&D Systems) was added to the mixture.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To elucidate the role of STAT5A in macrophage differentiation of
M1 cells, the wild-type STAT5A and the constitutively active mutant
STAT5A1*6 were transduced into M1 cells using a bicistronic retrovirus
vector, pMX-IRES-EGFP, and a retrovirus packaging cell line, BOSC23
(19). EGFP-positive M1 cells transduced with
pMX-STAT5A-IRES-EGFP and pMX-STAT5A1*6-IRES-EGFP were sorted on FACS
48 h after virus infection, as previously described
(21), and were termed M1/WT-STAT5A and M1/STAT5A1*6,
respectively. With May-Grünwald-Giemsa staining of cytospin
preparations, the sorted M1/WT-STAT5A and M1/STAT5A1*6 cells exhibited
the typical blast morphology of the parental M1 cells until 4 days
after virus infection (data not shown). In flow cytometric analysis,
both transfectants displayed little phenotypic change within 4 days,
although both expressed a high level of EGFP (Fig. 1
A). However, 7 days after the
virus infection, some of M1/STAT5A1*6 became enlarged, and at 2 wk
after the infection, M1/STAT5A1*6 cells exhibited a mixed pattern of
morphology in culture, with both nonadherent and adherent cells.
Microscopic examination of the M1/WT-STAT5A and the M1/STAT5A1*6 cells
following May-Grünwald-Giemsa staining showed that STAT5A1*6, but
not the wild-type STAT5A, induced M1 cells to differentiate into
various stages along the monocytic differentiation pathway, giving rise
to a heterogeneous population of blast, intermediate, and mature
monocytic stages (Fig. 1B). Flow cytometric analysis was
also done to quantitate the morphological changes in the M1/WT-STAT5A
and the M1/STAT5A1*6 cells. The increase in size and granulosity of the
cytoplasm, a hallmark of monocytic differentiation,
observed in M1/STAT5A1* 6 cells was evaluated by forward scatter
(FSC) and side scatter (SSC), respectively (Fig. 1C,
upper panels), and 66.4% of M1/STAT5A1*6 cells showed a
shift from region R1 to region R2 with increased size and granulosity.
This shift pattern is similar to that of parental M1 cells cultured in
the presence of IL-6 (100 ng/ml) for 5 days (data not shown). In
addition, most of M1/STAT5A1*6 cells exhibited de novo expression of a
myelomonocytic marker CD14 (Fig. 1D). IL-6, LIF, and human
OSM induce terminal differentiation of the parental M1 cells into
macrophages and eventuate cell death (13, 14, 15). In
contrast, M1/STAT5A1*6 cells underwent monocytic differentiation, with
a proliferating population retaining blastic phenotypes. Three such
clones were isolated by limiting dilution and were
termed MD1, MD2, and MD3 (M1 cells undergoing monocytic differentiation
with expression of the STAT5A1*6). Flow cytometric analysis of these
clones revealed basically similar phenotypes (Fig. 1E). They
continuously proliferated, retaining a population of blast (region R1)
while some acquired phenotypes of differentiated monocytes (region R2).
As shown in Fig. 2A, STAT5A
was constitutively phosphorylated on tyrosine residues in the MD1, MD2,
and MD3 cells (lanes 3-5). The EMSA of
these clones demonstrates that STAT5A in these cells showed DNA binding
activity (Fig. 2B), which was supershifted by the
anti-STAT5 Ab. Thus, STAT5A1*6 was constitutively activated in the
retrovirally transduced M1 cells and induced differentiation of M1
cells into a macrophage lineage.
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Using the M1 stable transfectants expressing STAT5A1*6 (MD1–3),
we attempted to clarify the molecular mechanism by which STAT5A1*6
induced macrophage differentiation of M1 cells. It was reported that a
dominant-negative form of STAT3 profoundly inhibited IL-6-induced
macrophage differentiation of M1 cells, and that STAT3 was likely to
play a central role in the differentiation of M1 cells
(23). In contrast, because IL-3 and GM-CSF are capable of
inducing granulocyte-macrophage colony formation in in vitro cultures
of bone marrow cells of normal mice (24), STAT5 may also
play a role in myelomonocytic development. We first asked whether STAT3
in MD cells was phosphorylated on tyrosine residues. Indeed, STAT3 was
phosphorylated on tyrosine residues in MD cells expressing STAT5A1*6
(Fig. 3A, lanes
3-5), but not in parental M1 cells or M1 cells
expressing the wild-type STAT5A (Fig. 3A, lanes 1
and 2). One possibility was that the constitutively active
STAT5A heterodimerized with STAT3. To test the possible interaction
between STAT5A1*6 and STAT3 in MD cells, we performed
immunoprecipitation using the anti-STAT5A Ab followed by
immumoblotting with the anti-STAT3 Ab and vice versa. However, we
did not detect a heterodimer of STAT5 and STAT3 in MD cells (Fig. 3B), indicating that STAT5A1*6 was capable of inducing
activation of STAT3 through mechanisms other than forming heterodimers
with STAT3.
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It was reported that IL-3 induced production of IL-6 in several
IL-3-dependent hemopoietic cells (25). Because
STAT5A is one of the key molecules that mediate IL-3-induced cell
signaling (26), and IL-6 induces STAT3 activation through
IL-6R (27, 28, 29), we hypothesized that STAT5A1*6 induces
macrophage differentiation of M1 cells through activation of IL-6
production. To test this hypothesis, we examined the supernatant of MD
cells for the ability to induce differentiation of parental M1 cells.
As shown in Fig. 4A, the
supernatant of the MD cells induced macrophage differentiation in
parental M1 cells, and an anti-IL-6 neutralizing Ab (10 µg/ml)
profoundly blocked this activity of the supernatant. The anti-IL-6
neutralizing Ab (10 µg/ml) also led to decreased tyrosine
phosphorylation of STAT3 in parental M1 cells (Fig. 4B). In
addition, IL-6 mRNA was readily detected in the MD cells, but not so in
parental M1 cells (Fig. 4C). Thus, STAT5A1*6 expressed in MD
cells produced a sufficient amount of IL-6 production to induce
macrophage differentiation of parental M1 cells via activation of
STAT3.
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Whereas the supernatant of the MD cells strongly induced terminal
differentiation and eventually apoptosis in parental M1 cells, the MD
cells proliferated continuously, with some populations having
differentiated phenotypes. To identify key molecules that inhibited MD
cells from apoptosis following terminal differentiation in the
supernatant containing high concentrations of IL-6, we generated a cDNA
library in a retrovirus vector pMXneo (30) from one of the
MD clones, MD1. Genes with the potential to inhibit apoptosis following
terminal differentiation were searched for in retrovirus-mediated
functional screening of the MD-1-derived cDNA library; M1 cells
infected with the cDNA library were screened for clones resistant to
IL-6-induced apoptosis in a medium containing IL-6 (100 ng/ml) and G418
selection reagents (600 µg/ml). A clone that survived and grew in the
presence of IL-6 was isolated after screening of 1.5 x
105 independent cDNA clones. The integrated cDNA
in the clone, recovered by PCR with vector primers, was sequenced. The
cDNA integrated in the clone was found to be a sense cDNA encoding the
full-length of A1, which is a member of the bcl-2 family of
proteins known to inhibit apoptosis (31, 32, 33). As shown in
Fig. 5A, the expression level
of A1 mRNA was extremely high in MD cells, but undetectable
in parental M1 cells. In addition, expression of A1 mRNA was
not detected when parental M1 cells underwent IL-6-induced terminal
differentiation and apoptosis (data not shown), which suggested that
A1 was one of the responsible genes for the resistance of MD
cells to IL-6-induced apoptosis following terminal differentiation. To
confirm that expression of the A1 gene protected M1 cells
from IL-6-induced apoptosis, A1 cDNA was subcloned into
pMX-IRES-EGFP and reintroduced into M1 cells. As a control,
pMX-IRES-EGFP was also introduced into M1 cells. EGFP-positive cells
were sorted on FACS 48 h after virus infection, as described in
Materials and Methods. Morphological changes of both M1
transfectants under the treatment with IL-6 (10 ng/ml) for 5 days are
shown in Fig. 5B. Consistent with an observation previously
reported (31), the constitutive expression of
A1 efficiently protected M1 cells from IL-6-induced
apoptosis. Therefore, STAT5A1*6 induced not only IL-6 production that
could induce macrophage differentiation of parental M1 cells, but also
expression of A1 that could counterbalance the effect of
IL-6 by protecting M1 cells from apoptosis following terminal
differentiation.
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To confirm that the autocrined IL-6 played a central role in
STAT5A1*6-induced macrophage differentiation of M1 cells, we cultured
the STAT5A1*6-transduced M1 cells in the presence of an anti-mIL-6
neutralizing Ab (10 µg/ml) immediately after the sorting of
EGFP-positive cells. The anti-IL-6 Ab profoundly inhibited the
differentiation of M1 cells expressing STAT5A1*6 (Fig. 6). We also confirmed that an isotype
control Ab did not affect this differentiation (data not shown). These
results clearly show that STAT5A1*6 induced differentiation of M1 cells
through autocrine production of IL-6.
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B p65
Next, we asked whether STAT5A directly activated the promoter of
the IL-6 gene, which does not contain the typical STAT5A binding site
(34). For this, we did a transactivation assay in which
Ba/F3 cells were cotransfected with IL-6 promoter-luciferase reporter
constructs harboring various deletions in the IL-6 promoter region
(Fig. 7A), and a vector
carrying the wild-type STAT5A or STAT5A1*6. As shown in Fig. 7B, the transactivating activity of pMX-STAT5A1*6 toward the
wild-type IL-6 promoter (k0) was stronger (
30-fold) than that of
pMX-STAT5A in the absence of IL-3. Using mutant constructs, mk9 and
mk0, harboring mutations in the NF-B binding site, it was also found
that the NF-B binding site was indispensable in the STAT5A-induced
transactivation of the IL-6 promoter. When Ba/F3 cells were treated
with IL-3 (1 ng/ml) after cotransfection with k0, the transactivating
activities of both pMX-STAT5A and pMX-STAT5A1*6 were more potent
(10-fold) than those without IL-3 treatment (Fig. 7C).
These results are consistent with our previous data describing the
transactivation potency of STAT5A1*6 (8). We also reported
that another STAT5A mutant, STAT5A-N642H, harboring a point mutation in
the Src homology 2 domain showed a phenotype identical with that of
STAT5A1*6 (9), indicating that the finding with STAT5A1*6
was not unique to a particular mutant. These data suggest that
activation of STAT5A by IL-3 induced transactivation of the IL-6
promoter through the NF-B binding site.
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B activation is involved in the STAT5A-mediated
transactivation of IL-6, we performed EMSA using COS cells after
transient gene expression of EpoR and STAT5A, as described in
Materials and Methods. As shown in Fig. 8, the DNA binding activity of NF-B
p65 in cells cotransfected with pMX-STAT5A1*6 (lane
5) was notably enhanced compared with those in cells cotransfected
with pMX and pMX-STAT5A (lanes 3 and 4).
When COS cells were stimulated with Epo after cotransfection, the DNA
binding activities of NF-B p65 in the cells were also enhanced
(lanes 2, 4, and 6) compared
with those without treatment of Epo (lanes 1,
3, and 5). The DNA-protein complexes were
confirmed to contain NF-B p65, but not STAT5A, using an
anti-NF-B p65 Ab (lanes 7–12) or an
anti-STAT5A Ab (data not shown). The DNA binding activity of
NF-B p50 was not affected by the coexpression of STAT5A without
stimulation (data not shown).
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B
in M1/WT-STAT5A and MD cells expressing the constitutively active STAT5
mutant STAT5A1*6. As shown in Fig. 9A, the DNA-protein complex
formation was specific to the oligonucleotides corresponding to the
NF-B site and enhanced in MD cells. In addition, the DNA-protein
complexes in MD cells showed a slower mobility than those in
M1/WT-STAT5A cells. These results suggest that the DNA-NF-B
complexes in MD cells were somehow modulated by STAT5A1*6 and formed
larger complexes that may contain additional molecule(s). Using the
anti-NF-B p65 Ab, we found that the lower band of the
DNA-NF-B complexes contained NF-B p65 (Fig. 9B). In
contrast, the upper band of the DNA-protein complexes in MD cells
contained c-Rel (Fig. 9C). These findings imply that
activation of STAT5A transactivated IL-6 expression not only by
enhancing the DNA binding activity of NF-B p65 and c-Rel, but also
by modulating the DNA-protein complexes of NF-B p65 in MD cells.
Using the anti-STAT5A Ab, we also performed EMSA in MD cells to
test whether this mobility shift was induced by the direct association
of STAT5A1*6 with the DNA-NF-B complexes. However, the DNA-NF-B
complexes were not supershifted by the anti-STAT5A Ab (data not
shown). We are now investigating how expression of STAT5A1*6 leads to
mobility shift of the DNA-protein complexes of NF-B p65 in MD
cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B p65 in inducing IL-6 production in M1
cells.
We first considered that p21WAF/Cip1 played
important roles in inducing M1 differentiation because
p21WAF/Cip1 was a target gene of STAT5
(25, 35, 36). In fact, overexpression of
p21WAF/Cip1 alone was able to induce partial
differentiation of Ba/F3 cells into macrophage-like cells
(10). However, this was not the case in MD cells;
STAT5A1*6 did not induce detectable levels of
p21WAF/Cip1 (data not shown). Instead, we found
that STAT5A1*6 transactivated the IL-6 promoter, and that produced
IL-6-induced STAT3 activation and macrophage differentiation in an
autocrine fashion. It is known that transactivation of the IL-6
promoter is regulated by NF-B, NF-IL-6, and AP-1 (37, 38), and that the promoter region contains no STAT5 binding
sites. Using deletion mutants of the IL-6 promoter, we found that the
NF-B binding site of the IL-6 promoter was sufficient for
STAT5A-induced transactivation of IL-6. Moreover, the DNA binding
activity of NF-B p65 was enhanced by STAT5A1*6. These results
indicate that the STAT5A-induced IL-6 production was mediated by
NF-B activation. This was analogous to the reported data that STAT6
and NF-B are directly associated and function in a synergistic
fashion (39). However, we were not able to demonstrate
direct interaction between STAT5 and NF-B p65 (data not shown). It
is possible that other transcription factors, such as CREB binding
protein/p300 or a protein induced by STAT5, mediate their interaction.
In addition to IL-6, LIF and OSM, other members of the IL-6 family
cytokines, are also produced from Ba/F3 cells in response to IL-3
stimulation, and the promoter region of OSM actually harbors STAT5
binding sites (36). These data suggest that OSM is the
ancestral gene of the IL-6 gene family, and that production of these
three cytokines is regulated essentially by the same mechanism,
althrough the promoter of OSM, but not those of IL-6 and LIF, carries
the STAT5 binding sites.
Our findings provide evidence for a novel capability of STAT5 to
cooperate with NF-B p65 in inducing production of IL-6, and this
notion is consistent with several observations reported by other
investigators. First, in a patient with follicular lymphoma, autologous
bone marrow transplantation with high-dose IL-3 treatment induced
terminal differentiation of the lymphoma cells into plasma cells
followed by a long-lasting remission. This phenomenon was preceded by
elevation of the serum level of IL-6 after the treatment with IL-3
(40). Second, the roles of NF-B in macrophage
differentiation have been reported (41, 42, 43). Activation of
IL-6 gene expression through NF-B and the roles of IL-6 in
macrophage differentiation were also reported (14, 37, 38). Finally, GM-CSF induces NF-B activation in some types of
hemopoietic cells (44, 45). We also found that IL-3
induced NF-B transactivation in a reporter assay in Ba/F3 cells
(Fig. 7
C). Therefore, we propose a novel signal cascade of
myelomonocytic development: IL-3 and/or
GM-CSF
STAT5NF-BIL-6
autocrinemacrophagedifferentiation.
Analysis of c-rel-/- mice revealed that
Rel/NF-B is a critical transactivator of the promoter of the
A1 gene (46). Although the promoter of
A1 does not contain the typical STAT5A binding site
(46), STAT5A1*6 strongly induced A1 expression
in M1 cells (Fig. 5B). As shown in Fig. 9C,
STAT5A1*6 apparently enhanced the DNA binding activity of c-Rel as well
as NF-B p65. Therefore, it is likely that STAT5A1*6 controlled
expression of the A1 gene as well as the IL-6
gene via enhanced binding of Rel/NF-B to the promoters. An
antiapoptotic activity of STAT5 in primary myeloid hemopoietic cells
has also been reported by Kieslinger et al. (47). They
demonstrated antiapoptotic activity of STAT5 through the
bcl-2 family gene, bcl-x, whose promoter contains
binding sites of STAT5.
In addition to the promoter of IL-6, STAT5A1*6 transactivated the HIV
promoter, which has the NF-B site but not STAT5 binding sites
(48) (T. Kawashima and T. Kitamura, unpublished
results). Thus, cooperation of STAT5 and NF-B may play a role
in manifesting pleiotropic functions of STAT5.
The finding that STAT5A activation stimulates autocrine production of IL-6 implies an interesting possibility that downstream signals of the IL-3 or the GM-CSF receptor by themselves may not be sufficient to stimulate terminal differentiation of hemopoietic progenitors. Additional signals mediated by autocrine cytokines, such as IL-6 induced by IL-3 and GM-CSF, may be required for terminal differentiation of progenitor cells into myelomonocytic lineages.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Toshio Kitamura, Division of Hematopoietic Factors, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. E-mail: kitamura{at}ims.u-tokyo.ac.jp
3 Abbreviations used in this paper: EPO, erythropoitin; OSM, oncostatin M; m, mouse; EGFP, enhanced green fluorescent protein; IRES, internal ribosomal entry site; FSC, forward scatter; SSC, side scatter.
Received for publication November 14, 2000. Accepted for publication July 19, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chain but is not essential for the proliferative signal transmission. Proc. Natl. Acad. Sci. USA 92:5482.-chain of the GM-CSF/IL-3/IL-5 receptors that are required for inducing differentiation or clonal suppression in myeloid leukaemic cell lines. EMBO J. 16:451.[Medline]
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B transcription factors are involved in normal erythropoiesis. Blood. 91:4136.B in human CD34+ bone marrow cell survival. Blood. 93:3302.This article has been cited by other articles:
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  T. Kawashima, Y. C. Bao, Y. Nomura, Y. Moon, Y. Tonozuka, Y. Minoshima, T. Hatori, A. Tsuchiya, M. Kiyono, T. Nosaka, et al. Rac1 and a GTPase-activating protein, MgcRacGAP, are required for nuclear translocation of STAT transcription factors J. Cell Biol., December 18, 2006; 175(6): 937 - 946. [Abstract] [Full Text] [PDF]
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T. Nakamura, R. Ouchida, T. Kodama, T. Kawashima, Y. Makino, N. Yoshikawa, S. Watanabe, C. Morimoto, T. Kitamura, and H. Tanaka Cytokine Receptor Common beta Subunit-mediated STAT5 Activation Confers NF-kappa B Activation in Murine proB Cell Line Ba/F3 Cells J. Biol. Chem., February 15, 2002; 277(8): 6254 - 6265. [Abstract] [Full Text] [PDF]
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