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Blood, Vol. 93 No. 12 (June 15), 1999:
pp. 4154-4166
Dominant Negative Mutants Implicate STAT5 in Myeloid Cell
Proliferation and Neutrophil Differentiation
By
Robert L. Ilaria Jr,
Robert G. Hawley, and
Richard A. Van Etten
From The Simmons Cancer Center, University of Texas Southwestern
Medical School, Dallas, TX; the Hematopoiesis Department, Holland
Laboratory, American Red Cross, Rockville, MD; and The Center for Blood
Research, Department of Genetics, Harvard Medical School, Boston, MA.
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ABSTRACT |
STAT5 is a member of the signal transducers and activation of
transcription (STAT) family of latent transcription factors activated
in a variety of cytokine signaling pathways. We introduced alanine
substitution mutations in highly conserved regions of murine STAT5A and
studied the mutants for dimerization, DNA binding, transactivation, and
dominant negative effects on erythropoietin-induced STAT5-dependent
transcriptional activation. The mutations included two near the
amino-terminus (W255KR AAA and
R290QQAAA), two in the DNA-binding domain
(E437EAA and V466VVAAA),
and a carboxy-terminal truncation of STAT5A (STAT5A/ 53C) analogous
to a naturally occurring isoform of rat STAT5B. All of the STAT mutant
proteins were tyrosine phosphorylated by JAK2 and heterodimerized with
STAT5B except for the WKR mutant, suggesting an important role for this
region in STAT5 for stabilizing dimerization. The WKR, EE, and VVV
mutants had no detectable DNA-binding activity, and the WKR and VVV
mutants, but not EE, were defective in transcriptional induction. The
VVV mutant had a moderate dominant negative effect on
erythropoietin-induced STAT5 transcriptional activation, which was
likely due to the formation of heterodimers that are defective in DNA
binding. Interestingly, the WKR mutant had a potent dominant negative
effect, comparable to the transactivation domain deletion mutant,
53C. Stable expression of either the WKR or 53C STAT5 mutants in
the murine myeloid cytokine-dependent cell line 32D inhibited both
interleukin-3-dependent proliferation and granulocyte
colony-stimulating factor (G-CSF)-dependent differentiation, without
induction of apoptosis. Expression of these mutants in primary murine
bone marrow inhibited G-CSF-dependent granulocyte colony formation in
vitro. These results demonstrate that mutations in distinct regions of
STAT5 exert dominant negative effects on cytokine signaling, likely
through different mechanisms, and suggest a role for STAT5 in
proliferation and differentiation of myeloid cells.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
SIGNAL TRANSDUCERS and activation of
transcription (STAT) family members comprise a group of latent
transcription factors involved in cytokine, hormone, and growth factor
signaling.1,2 STAT5, originally described as mammary gland
factor for its essential role in mediating prolactin-induced gene
expression,3 has been implicated in a diverse range of
signal transduction pathways induced by the interleukin-3
(IL-3)/granulocyte-macrophage colony-stimulating factor (GM-CSF)/IL-5
family,4-6 the IL-2/IL-7/IL-15 family,7-10 erythropoietin,9,11 thrombopoietin,12
granulocyte colony-stimulating factor (G-CSF),13,14
epidermal growth factor,15 leptin,16 and
insulin.17 STAT5 is expressed in a variety of tissues and, like other STAT family members, binds to specific
phosphotyrosine-containing sequences on cytoplasmic receptors via its
Src homology 2 (SH2) domain. STAT5 exists in two principal forms,
STAT5A and STAT5B, which are encoded by separate genes. Although they
differ in their C-terminal regions, STAT5A and STAT5B are highly
homologous with each other and across species.6,18 Upon
receptor activation, a highly conserved tyrosine near the
carboxy-terminus (C-terminus) of STAT5 becomes phosphorylated by Janus
kinase (JAK) family members, resulting in dissociation of STAT5 from
the receptor, and STAT5A and 5B homodimerization or heterodimerization
by mutual SH2 domain-phosphotyrosine interactions. STAT5 dimers then
translocate to the nucleus and bind DNA at specific sequences, inducing
gene expression.
The biological effects of activation of STAT5 are incompletely
understood. Female mice with homozygous inactivation of the stat5a gene exhibit a failure of postpartum mammary gland
differentiation and lactation,19 demonstrating that STAT5A
is required for mammopoiesis and lactogenic signaling. Given the
prominent activation of STAT5 in response to hematopoietic cytokines
such as erythropoietin and IL-3, it was anticipated that STAT5 might
also be required for aspects of hematopoiesis, but the
stat5a /,19
stat5b/,20 and
stat5a/
stat5b/ double knockout
mice21 show no overt defects in the blood system. Although
STAT5 is apparently not required for hematopoiesis, it may still play a
role in the antiapoptotic and proliferative responses to hematopoietic
cytokines. Thymocytes from stat5a/
stat5b/ double knockout mice fail to
proliferate in response to IL-2,21 whereas granulocytic
cells from stat5a/ mice have defects
in GM-CSF-induced proliferation.22 A mutant of STAT5B with
a large deletion in the C-terminus exhibited dominant negative effects
on STAT5-dependent transcription and a modest inhibitory effect on
IL-3-dependent proliferation in the murine B-lymphoid cell line
Ba/F3.23 Subsequently, STAT5A mutants with smaller
C-terminal truncations of a basic transactivation domain have also been
demonstrated to have dominant negative effects on STAT5-dependent
transcription,24,25 but stable expression of one of these
mutants in the IL-3-dependent myeloid cell line 32D did not affect
proliferation or survival.25 A recently described constitutively active mutant of murine STAT5 has been shown to partially substitute for IL-3 for survival and proliferation of Ba/F3
cells.26 However, because this mutant was originally
selected for on the basis of these properties, the involvement of
endogenous STAT5 in these processes remains unresolved.
STAT5 activation may also play a role in the pathogenesis of human
leukemia. Constitutive activation of diverse STAT proteins is found in
a variety of primary human leukemia cells.27,28 In
particular, prominent activation of STAT5 is consistently observed in
cell lines from patients with chronic myeloid leukemia (CML) and in
IL-3-dependent cell lines transformed by the BCR/ABL
oncogene,29-31 likely due to direct phosphorylation by the
Bcr/Abl tyrosine kinase.29 Because Bcr/Abl can substitute
for IL-3 for survival and growth of IL-3-dependent hematopoietic cell
lines32,33 and both IL-3 and Bcr/Abl activate STAT5, it is
plausible that STAT5 activation may contribute to the pathogenesis of
human CML.
Because deletion mutagenesis may have multiple effects on STAT
function, we introduced specific point mutations in highly conserved
regions of murine STAT5A and studied their effect on heterodimerization, DNA binding, and transactivation. STAT5A point mutants were also evaluated for their ability to exert a dominant negative effect on erythropoietin-induced STAT5-dependent
transcriptional activation. The point mutants chosen for study included
two located in the amino-terminus and two in the putative DNA-binding
domain. In addition, we generated a novel carboxy-terminal STAT5A
truncation mutant that is similar to a naturally occurring isoform of
rat STAT5B.34 Three of the STAT5A mutants function as
dominant negative mutants and interfere with STAT5-dependent
transcription, likely through different mechanisms. Another of the
mutations implicates the STAT5A amino terminus in stabilization of
SH2-dependent dimerization. Two of the mutants inhibited
IL-3-dependent proliferation and G-CSF-induced neutrophil
differentiation upon stable expression in the myeloid cell line 32D and
decreased G-CSF-dependent granulocytic colony formation after
transduction of primary murine bone marrow. These results suggest a
role for STAT5 in growth and differentiation of myeloid cells.
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MATERIALS AND METHODS |
Cells and cell culture.
293T cells were grown in Dulbecco's modified Eagle's
(DME) medium supplemented with 10% heat-inactivated fetal
calf serum, penicillin/streptomycin, 2 mmol/L glutamine, and
nonessential amino acids. 32D Cl3 cells35 were grown in
RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf
serum, penicillin/streptomycin, 2 mmol/L glutamine, and 10%
WEHI-3B-conditioned medium (WEHI-CM)36 as a source of
IL-3.
Construction of STAT5A mutants.
A hemagglutinin (HA) epitope tag was added to the
NH2-terminus of murine STAT5A subcloned in pUC19 (New
England Biolabs, Beverly, MA) by inverse polymerase chain reaction
(PCR) and verified by dideoxy DNA sequencing. Point mutations were then
introduced into HA-tagged murine STAT5A by enzymatic inverse PCR using
mutagenic primers containing a new Kpn I restriction
site.37 All point mutations involved substitution of the
respective amino acid with alanine. For STAT5A/WKR, codons 255-257 were
changed from TGG, AAG, and CGG to GCG; for STAT5A/RQQ, codons 290-292 were changed from CGG, CAG, and CAG to GCG; for STAT5A/EE, codons 437 and 438 were changed from GAG to GCG; and for STAT5A/VVV, codons
466-468 were changed from GTG, GTC, and GTT to GCG, GCC, and GCT,
respectively. Positive clones were identified by the creation of a new
Kpn I site and confirmed by DNA sequencing. The STAT5A mutants
were then digested with Kpn I, blunted with T4 DNA polymerase,
and self-ligated. Mutant clones were identified by loss of the
Kpn I site and restoration of the reading frame confirmed by
DNA sequencing. The STAT5A/ 53C mutant was generated by inverse PCR
using a mutagenic oligonucleotide introducing a stop codon (TGA)
immediately after the asparagine at amino acid number 740, along with a
new Kpn I restriction site. The resulting clone, lacking the
last C-terminal 53 amino acids of murine STAT5A, was identified by
digestion with Kpn I and confirmed by DNA sequencing. Wild-type
and mutant forms of STAT5A were subcloned into the vector pcDNA3
(Invitrogen, San Diego, CA) for transient expression studies.
Transient transfection and  -casein-luciferase transactivation
assay.
293T cells were transfected by a modified calcium phosphate protocol as
previously described.38 To assess tyrosine phosphorylation and heterodimerization, cells were cotransfected with 2.5 µg STAT5A wild-type or mutant, 2.5 µg STAT5B expression construct, and 5 µg
of pEFBos/JAK2. For transactivation assays, sets of 4 plates were
transfected with 0.25 µg of each STAT5A mutant, cotransfected with
0.5 µg of pEFBos/JAK2 or parental pEFBos; one set was transfected with 1.0 µg of a wild-type -casein luciferase reporter and the other was transfected with 1.0 µg of a mutant -casein luciferase construct with point mutations in both STAT5 binding
sites.39 Twenty-four hours posttransfection, cell lysates
were prepared and luciferase activity was determined using an AutoLumat
LB953 luminometer (EG&G Berthold/Wallac, Inc, Gaithersburg, MD), as described.40 To correct for differences in transfection
efficiency, all transfections included 0.1 µg of a human growth
hormone (hGH) expression plasmid, RSV-hGH. hGH levels in culture medium
from transfected cells were measured by radioimmunoassay (Nichols
Institute, San Juan Capistrano, CA), compared with a standard curve,
and this value was used to normalize the level of luciferase activity between different transfections. To assess dominant negative effects, cells were cotransfected with 1.0 µg of pXM190 murine erythropoietin receptor expression construct, 0.25 µg of wild-type STAT5A , and either 1.0 or 2.0 µg of STAT5A mutant, along with the wild-type -casein luciferase reporter and RSV-hGH expression construct for
normalization. Twenty-four hours posttransfection, the cells were
stimulated with 10 U/mL recombinant human erythropoietin (Amgen,
Thousand Oaks, CA). Cell lysates were prepared 7 to 8 hours
poststimulation, and luciferase activity was measured and normalized as
described above.
Immunoprecipitation and Western blot.
Lysates in RIPA buffer were prepared as previously
described41 from approximately 1 × 107
transfected 293T cells or 32D cells stably expressing mutant STAT5A.
32D cells were starved of IL-3 for 3 hours and then stimulated with
10% (vol/vol) WEHI-CM or 100 ng/mL recombinant human G-CSF (Amgen) for
15 minutes at 37°C. Protein lysates, normalized by OD595 (Bio-Rad Protein assay; Bio-Rad Laboratories,
Hercules, CA), were subjected to immunoprecipitation by antisera to
STAT3, 5A, or 5B (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-HA monoclonal antibody (Berkeley Antibody Co, Richmond, CA) for 4 to 6 hours, were resolved by 5% to 9% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and were
electrophorectically transferred to a nitrocellulose membrane. Western
blot was performed with either anti-HA, anti-STAT, or
anti-phosphotyrosine (4G10; Upstate Biotechnology, Lake Placid, NY)
antibody and detected by enhanced chemiluminescence (Amersham,
Arlington Heights, IL).
Electromobility shift assay (EMSA).
Nuclear extracts were prepared from 293T cells transfected with either
wild-type or mutant STAT5A, with or without JAK2, as previously
described.42 Six micrograms of nuclear extract protein was
incubated with 0.2 ng of a 32P-labeled double-stranded
Fc RI GAS-based oligonucleotide probe29,43 for 20 minutes
at room temperature. Supershift analysis was performed by incubating
nuclear extracts with 1:15 dilutions of either anti-STAT5A (Santa Cruz
Biotechnology) or anti-HA (Berkeley Antibody Co) antibody for 30 minutes at 4°C. The DNA-protein complexes were resolved on 4%
tris-acetate-EDTA (TAE) polyacrylamide gels and detected by autoradiography.
Expression of dominant-negative STAT5A mutants in 32D cells.
The HA-tagged STAT5A/WKR and STAT5A/53C mutants were cloned into the
5' position of MINV, an MSCV-based retroviral vector containing
an internal ribosome entry site from encephalomyocarditis virus between
a 5' multiple cloning site and a 3' neomycin resistance gene.44 Helper-free high-titer retroviral supernatant was
prepared using the kat transient transfection
system,45 as described.38 32D cells were
transduced with retroviral stocks of the two STAT5 mutants and a vector
expressing the neomycin resistance gene alone by 24 hours of
cultivation in the presence of virus-containing supernatant, WEHI-CM,
and 6 µg/mL polybrene. Transduced cells were selected 24 hours
posttransduction in 1.0 mg/mL G418 (Geneticin; Life Technologies, Grand
Island, NY) and IL-3, and the G418-resistant populations were used for
studies of gene induction, proliferation, and differentiation
immediately thereafter.
RNA isolation and Northern blot.
G418-resistant populations of 32D cells transduced with the
neomycin-containing virus or with STAT5A/WKR and STAT5A/53C mutants were starved of IL-3 for 6 hours and then stimulated with 10% WEHI-CM
as a source of IL-3. RNA was prepared as described41,46 from 1 × 107 cells at 0, 30, 60, and 120 minutes
poststimulation; fractionated by formaldehyde agarose gel
electrophoresis; transferred to nylon membranes by capillary blot; and
probed with radioactive probes from the CIS, PIM-1, and
OSM genes.23
Assessment of IL-3-mediated proliferation and G-CSF-induced
differentiation.
Within 96 hours of retroviral transduction, G418-resistant populations
of 32D cells transduced with neomycin-containing virus or with the
STAT5A/WKR and STAT5A/53C mutants were seeded in duplicate at 5 × 105 cells in 10 mL of medium containing 10%
WEHI-CM but without G418 in a T-25 flask. Viable cells were counted
daily by trypan blue exclusion. For some time points, genomic DNA was
isolated from populations and DNA fragmentation was assessed by agarose
gel electrophoresis to exclude apoptosis. For G-CSF-induced neutrophil differentiation, cells were washed twice in phosphate-buffered saline
(PBS) and resuspended in medium containing 0.25% (vol/vol) WEHI-CM and
100 ng/mL recombinant human G-CSF (Amgen). Aliquots were removed for
fluorescence-activated cell sorting (FACS) analysis or cytospin and
Wright/Giemsa staining every 24 hours. FACS analysis was performed by
staining 105 fresh cells with biotinylated rat antimouse
CD11b (clone M1/70) or a biotinylated rat IgG2b/ isotype control
antibody (both from PharMingen, San Diego, CA), followed by
phycoerythrin-conjugated streptavidin. Cells were analyzed on a FACScan
flow cytometer (Becton Dickinson, San Jose, CA) with CellQuest software.
Retroviral transduction of primary murine bone marrow and
granulocyte colony-forming unit (CFU-G) analysis.
Bone marrow was harvested from 8- to 10-week old Balb/c mice (Taconic
Farms, Germantown, NY) and prestimulated47 for 24 hours in
medium containing DME, 15% (vol/vol) inactivated fetal calf serum, 5%
(vol/vol) WEHI-3B conditioned medium, penicillin/streptomycin, 1.0 µg/mL ciprofloxicin, 200 µmol/L L-glutamine, 6 ng/mL recombinant murine IL-3, 10 ng/mL recombinant murine IL-6, and 70 ng/mL recombinant murine stem cell factor (SCF; all from PeproTech, Rocky Hill, NJ). For
transduction, the three retroviral stocks were normalized by
appropriate dilutions to a titer of 1 × 107
G418-resistant CFU/mL, assessed on NIH 3T3 cells. After prestimulation, equal numbers of viable cells were transduced with either
MINV/STAT5A/WKR, MINV/STAT5A/53C, or MINV retrovirus alone in the
presence of 10 mmol/L HEPES, pH 7.4, and 2 µg/mL polybrene. To
increase transduction efficiency,48 the cells were then
cocentrifuged with virus at 1,000g for 90 minutes in a Sorvall
RT7 centrifuge, and the medium was changed after a 2- to 4-hour
adsorption period. Sixteen to 18 hours later, a second round of
retroviral transduction with cocentrifugation was performed, and the
cells were allowed to recover for 4 to 6 hours at 37°C. Cells were
then removed, washed twice in PBS, and plated in triplicate at a
density of 1 × 105 cells per 3.5-cm plate in
Methocult M3230 medium (StemCell Technologies, Vancouver, British
Columbia, Canada) containing 1 mg/mL (absolute) G418 and 100 ng/mL
recombinant human G-SCF. To assure that colonies were truly
G-CSF-dependent, cells were also plated in the same medium lacking
G-CSF. The number of G-CSF-dependent colonies was scored on day 4 to 5 and granulocytic morphology was confirmed by cytospin.
| |
RESULTS |
The STAT5A mutants are tyrosine phosphorylated by JAK2 and all
heterodimerize with STAT5B except for STAT5A/WKR.
To assess the impact of STAT5 mutagenesis on heterodimerization,
tyrosine phosphorylation, DNA binding, and transcriptional activation,
alanine substitution mutations were introduced into murine STAT5A in
regions highly conserved among STAT family members (Fig 1). Two point mutants were located
near the NH2-terminus, designated STAT5A/WKR
(W255KRAAA) and STAT5A/RQQ
(R290QQAAA), and two were located in the putative
DNA-binding domain, STAT5A/EE (E437EAA) and
STAT5A/VVV (V466VVAAA). An HA epitope tag was added at the NH2-terminus to distinguish mutant from
wild-type STAT5A. A C-terminal truncation mutant was also constructed
by placement of a stop codon, resulting in deletion of the last 53 amino acids (STAT5A/53C), analogous to a naturally occurring rat
STAT5B isoform.34
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| Fig 1.
Mutagenesis of conserved regions of murine STAT5A. Amino
acid sequence alignment of the central region of several mammalian (H,
human; M, mouse; R, rat) STAT family members is shown. Numbering is
from the amino terminus, and alignment was performed by the program
PRETTY.PILEUP (GCG, University of Wisconsin). The consensus sequence is
given at the bottom; a capital letter indicates a highly conserved
amino acid (at least 8 of 12 members). The numbering at the top of each
panel refers to the consensus sequence and because of gaps in the
homology is not correct for STAT5A. The positions of the canonical Src
homology 2 (SH2) domain and the putative DNA-binding domain as defined
by Horvath et al57 are indicated by the shaded boxes,
whereas the conserved JAK phosphorylation site is indicated by the
arrowhead. The locations of the alanine substitution mutations
WKRAAA, RQQAAA, EEAA, and
VVVAAA are indicated by bold face A characters below the
sequence. The positions of insertion of termination codons in the
murine STAT5A C-terminal truncation mutants 53C (this report),
683,23 749,24 713,25 and
65049 are shown by arrowheads.
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The expression and biological properties of these mutants were assessed
in 293T cells (Table 1), which were chosen
because of their lack of endogenous STAT5A (data not shown) and high
transfection efficiency. All of the STAT5A mutants were readily
expressed and, as expected, comigrated with wild-type STAT5A, except
for the truncation mutant STAT5A/53C
(Fig 2, top panel). Transfection of
wild-type STAT5A alone did not result in any tyrosine phosphorylation of STAT5, due to the very low level of endogenous JAK2 (data not shown). Therefore, to evaluate activation of mutant or wild-type STAT5A, 293T cells were cotransfected with or without supplemental JAK2
and analyzed by anti-HA immunoprecipitation and anti-phosphotyrosine Western blot. All of the STAT5A mutants were tyrosine phosphorylated in
the presence of JAK2 (Fig 2, middle panel), although the WKR and VVV
mutants appeared to be phosphorylated at lower levels than the other
proteins. Because 293T cells contain low endogenous levels of STAT5B
(data not shown), to assess STAT5 heterodimerization, 293T cells were
transfected with HA-tagged mutant or wild-type STAT5A, together with
STAT5B and JAK2, and analyzed for the ability of the STAT5A mutants to
coimmunoprecipitate STAT5B. STAT5A wild-type, STAT5A/VVV, STAT5A/EE,
and STAT5A/RQQ formed heterodimers with STAT5B in a JAK2-dependent
manner (Fig 2, bottom panel). Although STAT5A/WKR was tyrosine
phosphorylated by JAK2, it failed to heterodimerize with STAT5B,
suggesting that this region of STAT5A may be important in stabilizing
STAT dimerization. The C-terminal truncation mutant, STAT5A/53C,
also retained the ability to heterodimerize with STAT5B, confirming
that the transactivation domain is not required for dimerization.
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| Fig 2.
The STAT5A mutants are tyrosine-phosphorylated by JAK2,
but the STAT5A/WKR mutant fails to heterodimerize with STAT5B.
Wild-type or mutant STAT5A was coexpressed in 293T cells with STAT5B
with or without JAK2; in the left two lanes, STAT5B alone was expressed
with and without JAK2. Proteins were immunoprecipitated with anti-HA
( -HA) or anti-STAT5B (-5B) antibodies and blotted with anti-HA
(top panel), anti-phosphotyrosine (middle panel), or anti-STAT5B
(bottom panel) antibodies. Note that the anti-STAT5B antibody also
recognizes STAT5A and the STAT5A mutants with the exception of 53C,
which is lacking the C-terminal epitope recognized by this
antisera. The positions of the STAT5A and STAT5B proteins are
indicated by the arrowheads at right. The STAT5A point mutants (5A-PM)
migrate more slowly than STAT5B due to additional amino acids at the
C-terminus and the HA epitope tag at the N-terminus. When coexpressed
with JAK2, STAT5B migrates as three distinct forms (left 2 lanes,
bottom panel), of which the two most slowly migrating forms are
tyrosine phosphorylated (5B-PY, middle panel).
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All STAT5A point mutants lack GAS DNA-binding ability except for
STAT5A/RQQ.
The DNA-binding ability of the STAT5A mutants was assessed by EMSA
using a GAS probe derived from the FcRI gene
promoter.29,43 Nuclear extracts were prepared from 293T
cells transfected with wild-type or mutant STAT5A in the presence or
absence of JAK2. Transfection of 293T cells with JAK2 alone, or
wild-type or mutant STAT5A in the absence of exogenous JAK2, resulted
in no detectable GAS DNA-binding activity
(Fig 3). STAT5A-specific DNA binding was
confirmed by supershift of the DNA-protein complex with either anti-HA
(data not shown) or anti-STAT5A antibody. The point mutant STAT5A/RQQ
exhibited GAS DNA binding similar to wild-type STAT5A, whereas
STAT5A/53C demonstrated a DNA-protein complex even more prominent
than parental STAT5A, consistent with the observation that the
transactivation domain may negatively regulate STAT5A DNA
binding.24 In contrast, the point mutants STAT5A/VVV and STAT5A/EE demonstrated no appreciable GAS DNA-binding activity, demonstrating the importance of these highly conserved regions in the
STAT5A DNA-binding domain. STAT5A/WKR also had no detectable GAS
DNA-binding activity, likely because of its inability to homodimerize or heterodimerize. Similarly, a STAT5A mutant with a large truncation of the C-terminus, STAT5A/650,49 also did not bind DNA,
likely due to the absence of the Y694 JAK2 phosphorylation site that is
required for dimerization.
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| Fig 3.
The STAT5A/WKR, EE, and VVV mutants lack FcRI GAS
DNA-binding activity. Nuclear extracts were prepared from 293T cells
transfected with JAK2 and the indicated murine STAT5A mutants and
analyzed by EMSA using a 32P-labeled FcRI-derived GAS
probe. 293T cells transfected with either parental STAT5A (5A) alone or
JAK2 (JK2) alone are shown at left. STAT5-specific GAS DNA-binding
activity was confirmed by incubation with anti-STAT5A antibody
(5A+JK2+AB). The positions of the STAT5 GAS DNA-binding complex
(lower arrowhead) and the supershifted complex (upper arrowhead) are
indicated. Similar supershifts using anti-HA antibody were observed for
the STAT5A/RQQ and STAT5A/53C complexes (data not shown).
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STAT5A/VVV and STAT5A/WKR are defective in transcriptional activation
of a -casein-luciferase reporter.
The transactivation ability of wild-type and mutants STAT5A proteins
was assessed using a construct containing a minimal cytomegalovirus (CMV) promoter and tandem -casein DNA elements upstream
of a luciferase reporter gene.39 293T cells were
transfected with this reporter and each of the STAT5A mutants with or
without JAK2, and the level of transactivation was compared with
wild-type STAT5A by quantitation of luciferase activity in cell
extracts. A parallel experiment using a -casein luciferase reporter
with point mutations in the STAT5 binding sites39 was used
to control for nonspecific transactivation. None of the STAT5A species
caused significant transcriptional activation of the -casein
luciferase reporter in the absence of JAK2
(Fig 4). When coexpressed with JAK2,
wild-type STAT5A induced a fourfold activation of the luciferase
reporter, whereas the carboxy-terminal truncation mutant STAT5A/650
completely lacked transactivation ability. Although STAT5A/53C
demonstrated a significant decrease in -casein transcriptional
activation, it retained some transactivation ability, suggesting that
this deletion did not eliminate the entire STAT5A transactivation
domain. The VVV and WKR point mutations completely abolished STAT5A
transactivation ability, reflecting their potent inhibitory effect on
DNA binding. The STAT5A/RQQ mutant, on the other hand, demonstrated
little impairment in transactivation, consistent with its preserved
DNA-binding ability and intact transactivation domain. Interestingly,
STAT5A/EE exhibited relatively normal -casein transcriptional
activation despite the lack of DNA-binding activity by EMSA. None of
the STAT5A proteins significantly stimulated transcription of the mutant -casein luciferase reporter, demonstrating that
transcriptional activation in this assay is dependent on STAT5 binding
sites in the promoter.
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| Fig 4.
The STAT5A/VVV and STAT5A/WKR mutants are defective in
transcriptional activation. 293T cells were transfected with pcDNA3
vector alone, wild-type STAT5A (WT), or the indicated STAT5A mutant in
the absence ( ) or presence (+) of JAK2 and either wild-type ( )
or point mutant ( ) -casein luciferase reporter. The
transcriptional activation of the mutant or wild-type -casein
reporter was assessed by luciferase assay and expressed in relative
light units (RLU). To control for variations in transfection
efficiency, all samples were cotransfected with an hGH expression
plasmid to allow normalization of RLU values to secreted levels of hGH,
determined by radioimmunoassay. Error bars represent the standard error
based on at least two independent experiments.
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The point mutants STAT5A/WKR and STAT5A/VVV and the transactivation
domain deletion mutant STAT5A/53C exert a dominant negative effect
on erythropoietin-induced STAT5-dependent transcriptional activation in
293T cells.
Our STAT5A mutants demonstrated defects in various aspects of STAT5A
function, including dimerization, DNA binding, and transactivation. To
determine whether any of these mutants could interfere with wild-type
STAT5 signaling, an erythropoietin receptor expression system was used.
293T cells (which lack erythropoietin receptors) were transfected with
wild-type STAT5A, murine erythropoietin receptor, and each of the
STAT5A mutants, and dominant negative activity was assessed by
inhibition of erythropoietin-induced activation of the casein
luciferase reporter (Fig 5). Neither wild-type nor mutant STAT5A demonstrated significant activation of the
reporter in the absence of erythropoietin (data not shown). In the
absence of any STAT5A mutant, erythropoietin stimulation resulted in
agreater than fivefold increase in -casein luciferase reporter
activity. Addition of a dominant negative JAK2 mutant (DNJAK/829)50 completely blocked this effect. Consistent
with a recent report, the mutant STAT5A/53C, lacking most of the
STAT5A transactivation domain,24 severely inhibited
-casein reporter activity. The point mutant STAT5A/WKR also
demonstrated a potent dominant negative effect, similar to the 53C
mutant. The other four STAT5A mutants were intermediate in their
ability to block transactivation by wild-type STAT5A. STAT5A/650 and
STAT5A/VVV exhibited dose-dependent dominant negative activity
consistent with their lack of intrinsic transcriptional activation and
with previous observations.49 The STAT5A/RQQ or STAT5A/EE
mutants decreased transactivation by wild-type STAT5A, but this
inhibitory activity was not dose-dependent, suggesting these latter
mutants do not function significantly as dominant negatives, consistent with their preserved transactivation ability.
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| Fig 5.
The STAT5A/WKR, STAT5A/VVV, and STAT5A/53C mutants
exert dominant negative effects on erythropoietin-induced,
STAT5-dependent transcription. The indicated STAT5A mutant in the
expression vector pcDNA3 ([] 1 µg; [] 2 µg) was
cotransfected into 293T cells together with wild-type STAT5A plasmid
(0.25 µg), a murine erythropoietin receptor expression construct,
-casein luciferase reporter, and hGH expression plasmid as an
internal control. Cells were stimulated with erythropoietin as
described in Materials and Methods, and the increase in luciferase
activity relative to unstimulated cells, normalized for transfection
efficiency, is shown. Omission of the erythropoietin receptor
expression construct from the transfection resulted in background
levels of erythropoietin-stimulated luciferase activity in transfected
cells (data not shown). pcDNA3 indicates cotransfection with vector
alone. Error bars represent the standard error based on at least two
independent experiments.
|
|
STAT5A/WKR expression in 32D cells inhibits the activation of the
IL-3-responsive genes cis, pim-1, and osm.
To confirm a dominant negative effect on transcription in hematopoietic
cells, STAT5A/WKR was expressed in the murine hematopoietic factor-dependent cell line 32D and activation of the IL-3-responsive genes cis, pim-1, and osm23 was
analyzed by Northern blot. Control 32D/Neo cells showed evidence of
cis and pim-1 gene activation as early as 30 minutes
after IL-3 stimulation (Fig 6 and data not
shown), with a maximum transcriptional induction at 120 minutes. In 32D
cells expressing STAT5A/WKR, cis gene activation was severely impaired, with cis mRNA levels remaining at barely detectable levels for up to 120 minutes after IL-3 stimulation (Fig 6, upper panel). Pim-1 gene transcription was also suppressed in
STAT5A/WKR-expressing cells, with only a slight increase at 60 minutes
after IL-3 stimulation (Fig 6, middle panel). In 32D/Neo cells,
osm gene transcription peaked at 30 minutes and was readily
detectable for up to 2 hours; however, in 32D cells expressing
STAT5A/WKR, osm mRNA levels only reached barely detectable
levels 30 minutes after IL-3 stimulation and were undetectable
thereafter (Fig 6, lower panel). Similar results were also observed
upon expression of the STAT5A/53C mutant (data not shown).
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| Fig 6.
STAT5A/WKR inhibits the expression of the
IL-3-responsive genes pim-1, cis, and osm in
the myeloid cell line 32D. 32D cells expressing either the neomycin
resistance gene alone (32D/Neo) or STAT5A/WKR (32D/WKR) were deprived
of IL-3 for 6 hours and then stimulated with 10% WEHI-3B-conditioned
medium. Total RNA was prepared at the indicated time periods and
analyzed by Northern blot using 32P-labeled PIM-1,
CIS, or OSM probes. All lanes had equal loading as
judged by ethidium bromide staining of ribosomal RNAs (data not
shown).
|
|
32D cells expressing either STAT5A/WKR or STAT5A/53C demonstrate
decreased IL-3-dependent proliferation in vivo.
Because STAT5A/WKR and STAT5A/53C exerted dominant negative effects
on STAT5-dependent erythropoietin-induced transcriptional activation
and the activation of several IL-3-responsive genes, further studies
were performed to determine their impact on IL-3-dependent proliferation. Because of the possibility that dominant negative STAT5
expression might have deleterious effects on proliferation, STAT5A/WKR
and STAT5A/53C were introduced into 32D cells by retroviral gene
transfer (with an transduction efficiency of 25% to 30%; data not
shown) and proliferation assays were performed immediately after
selection in G418. Compared with 32D cells transduced with virus
carrying the neomycin resistance gene alone, cells expressing STAT5A/WKR demonstrated a moderate decrease in proliferation in 10%
WEHI-3B-conditioned medium (Fig 7). There
was no evidence of apoptosis in STAT5A/WKR-expressing 32D cells, even
when grown in minimal (0.25% WEHI-CM) amounts of IL-3 (data not
shown). Western blot analysis of the G418-resistant 32D-STAT5A/WKR
population confirmed expression of STAT5A/WKR at levels about threefold
higher than endogenous STAT5A when compared with neo-transduced cells (data not shown). STAT5A/53C also exerted a dominant negative effect
on IL-3-dependent 32D cell proliferation (Fig 7), again without
evidence of apoptosis (data not shown).
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| Fig 7.
Expression of either STAT5A/WKR or STAT5A/53C in 32D
cells inhibits IL-3-dependent proliferation. 32D cells expressing the
neomycin resistance gene alone (32D/Neo), STAT5A/WKR (32D/WKR), or
STAT5A/53C (32D/53C) were grown in 10% WEHI-3B-conditioned
medium without G418 and viable cell number was determined daily. Data
points represent the average of at least two independent experiments
performed in duplicate and error bars represent the calculated standard
error. The day-2 cell number for 32D/Neo cells was significantly higher
than that of either 32D/WKR or 32D/53C (P < .01, t-test).
|
|
STAT5A/WKR and STAT5A/53C block the G-CSF-dependent
differentiation of 32D cells.
In addition to IL-3-dependent growth, 32D cells differentiate into
mature neutrophils upon treatment with exogenous G-CSF.51 Because STAT5 has been shown to be activated by G-CSF
treatment,13,14 the effect of dominant negative STAT5
expression on the G-CSF-induced differentiation of 32D cells was
evaluated. 32D cells expressing neomycin resistance alone, STAT5A/WKR,
or STAT5A/53C were stimulated with 100 ng/mL recombinant human G-GSF
in the presence of minimal IL-3 (to prevent apoptosis) and were
examined for morphological evidence of myeloid differentiation. 32D
cells transduced with the neomycin vector alone (32D/Neo)
differentiated into neutrophils by day 7 of G-CSF treatment, whereas
the 32D cells expressing the STAT5A/WKR and STAT5A/53C mutants
showed only limited morphologic maturation
(Fig 8A). When the number of mature and
undifferentiated cells were quantitated, 32D/STAT5A/WKR cells
demonstrated an approximately sixfold decrease in the number of
neutrophils and band forms compared with 32D/Neo cells (Fig 8B).
Virtually all 32D/Neo cells exhibited morphologic differentiation by
day 7, whereas approximately 65% of 32D/STAT5A/WKR cells remained
undifferentiated, resembling non-G-CSF-treated 32D cells. Consistent
with a delay in morphologic maturation, 32D/STAT5A/WKR cells also
showed a higher percentage of myelocytes and metamyelocytes compared
with 32D/Neo cells (22% and 16%, respectively). G-CSF-dependent
induction of the myeloperoxidase gene, another molecular marker of
neutrophil differentiation, was intact in 32D/WKR- and
32D/53C-expressing cells but delayed by about 1 day relative to
32D/Neo cells (data not shown), indicating that STAT5 is not required
for some transcriptional responses in the maturation pathway induced by
G-CSF. Coincident with the block in morphological neutrophil
maturation, induction of the myeloid cell surface antigens Mac-1
(CD11b; Fig 8C) and Gr-1 (Ly-6G; data not shown) was inhibited to a
similar extent in 32D/WKR cells relative to 32D/Neo cells.
Overexpression of wild-type STAT5A in 32D cells had no effect on
proliferation or differentiation (data not shown). These results
suggest that STAT5 contributes to proliferation and is required for
neutrophil differentiation in 32D cells.
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| Fig 8.
STAT5A/WKR and STAT5A/53C block G-CSF-induced
neutrophil differentiation of 32D cells. (A) 32D cells expressing
either the neomycin resistance gene alone (32D/Neo), STAT5A/WKR
(32D/WKR), or STAT5A/53C (32D/53C) were placed in 100 ng/mL human
recombinant G-CSF and minimal IL-3 to permit cell viability throughout
the differentiation assay. Wright-Giemsa-stained cytospins were
prepared on the indicated days to assess G-CSF-dependent morphologic
maturation of 32D cells. The photographs depicted are representative of
at least two independent experiments. (B) Cytospins from day 7 of G-CSF
treatment from 32D/Neo and 32D/WRK cells were scored for neutrophil
differentiation on the basis of morphology, and the results of three
differential counts of 100 cells are each depicted with standard error.
(C) 32D/Neo and 32D/WKR cells were analyzed by FACS after staining with
anti-CD11b monoclonal antibody (thick line) or isotype control antibody
(thin line) on the day before (day 0) and the indicated days after
initiation of G-CSF treatment.
|
|
STAT5A/WKR and STAT5A/53C do not interfere with G-CSF-induced
STAT3 activation in 32D cells.
Expression of a dominant negative allele of STAT3 in a murine myeloid
cell line has been shown to block G-CSF-induced neutrophil maturation.52 To exclude the possibility that our dominant
negative STAT5 mutants might block neutrophil differentiation through
inhibition of STAT3, we examined STAT3 activation in 32D cells stably
expressing the dominant negative STAT5A mutants
(Fig 9). STAT3 was activated by G-CSF but
not IL-3 in parental 32D/Neo cells, and there was no appreciable
decrease in G-CSF-induced tyrosine phosphorylation of STAT3 in 32D/WKR
and 32D/53C cells (Fig 9, top panel). In parental 32D/Neo cells,
STAT5 was activated prominently by IL-3 and to a lesser extent by G-CSF
(Fig 9, bottom panel), as reported.13,14 Interestingly, in
32D/WKR cells, there was somewhat increased tyrosine phosphorylation of
STAT5 in response to both IL-3 and G-CSF, likely from phosphorylation
of the overexpressed STAT5A/WKR protein, which is recognized by the
anti-STAT5A antisera and comigrates with endogenous STAT5. In
32D/53C cells, there was decreased tyrosine phosphorylation of STAT5
in response to both cytokines, likely due to specific interference from
the truncated STAT5A/53C protein, which is not immunoprecipitated by
the anti-STAT5A antisera. Although anti-HA immunoprecipitates confirmed
expression of both dominant negative STAT5 proteins in the respective
cell lines, immunoprecipitation with this antibody was inefficient and
cytokine-induced tyrosine phosphorylation of the mutant STATs was not
detected under these conditions (data not shown). These results
demonstrate that the dominant negative effects of the STAT5A mutants
are restricted to STAT5 and do not interfere with activation of STAT3,
implying that STAT5 also plays an essential role in the differentiation of neutrophils.
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| Fig 9.
The STAT5A/WKR and 53C mutants do not inhibit STAT3
activation. Populations of 32D cells expressing either the neomycin
resistance gene alone (32D/Neo), STAT5A/WKR (32D/WKR), or STAT5A/53C
(32D/53C) were starved of IL-3 and then stimulated with IL-3 (I),
G-CSF (G), or unstimulated (NS). Extracts were precipitated with
anti-STAT3 (top 2 panels) or anti-STAT5A (bottom 2 panels) antibodies,
blotted to nitrocellulose, and hybridized with anti-phosphotyrosine
antibody (top panel of each pair). Membranes were then stripped and
reprobed with anti-STAT3 or anti-STAT5A antibodies, respectively
(bottom panel of each pair). Expression of STAT5A/53C (not
recognized by STAT5A antibody) was confirmed by anti-HA Western blot
(data not shown).
|
|
STAT5A/WKR and STAT5A/53C inhibit G-CSF-dependent granulocytic
colony formation in primary murine bone marrow.
Although 32D cells are frequently used to model granulocyte
development, a cell line may not accurately reflect normal myeloid cell
maturation. Therefore, we introduced the STAT5A/WKR and STAT5A/53C mutants into primary murine bone marrow by retroviral transduction and
analyzed their effect on G-CSF-dependent granulocytic colony formation
in vitro. Primary marrow cells were transduced with parental virus
(MINV) expressing neomycin resistance gene alone and with viruses
coexpressing either STAT5A/WKR or STAT5A/53C with the neomycin
resistance gene via an internal ribosome entry site (MINV/WKR and
MINV/53C, respectively). After plating in semisolid medium
containing G418 and G-CSF, we observed a 40% to 60% reduction in
granulocytic colony formation after transduction with the DN-STAT5A
mutants, relative to vector-transduced cells (Fig 10). No colonies were observed in
mock-infected cultures or in the absence of G-CSF (data not shown).
These results suggest that STAT5 contributes to granulocytic
differentiation during normal myelopoiesis.
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| Fig 10.
The STAT5A/WKR and 53C mutants inhibit
G-CSF-dependent granulocytic colony formation in primary murine bone
marrow. Primary murine bone marrow was transduced with parental MINV
retrovirus (MINV) or MINV retrovirus expressing STAT5A/WKR (MINV/WKR)
or STAT5A/53C (MINV/53C) and then plated in triplicate in
methylcellulose culture with G418 and exogenous G-CSF. The mean number
of granulocytic colonies on day 4 to 5 per 105 cells plated
is shown and is representative of three independent experiments. Error
bars indicate the standard error. No colonies were observed in
sham-infected cultures selected in G418 or upon omission of G-CSF from
the medium.
|
|
| |
DISCUSSION |
We have introduced point mutations in highly conserved regions of
murine STAT5A to gain insight into their impact on STAT5 heterodimerization, tyrosine phosphorylation, DNA binding, and transactivation. Two point mutants in the amino-terminal region of
STAT5A, STAT5A/WKR and STAT5A/RQQ, and two in the DNA-binding domain,
STAT5A/EE and STAT5A/VVV, were chosen for study. Because deletions in
the transactivation domain of STAT5A have been demonstrated to
interfere with wild-type STAT5A signaling,24 a novel
carboxy-terminal truncation mutant, STAT5A/53C, was also constructed.
The STAT amino-terminal domain is a highly conserved region that may
play a role in signaling specificity and regulation. Recently, a
deletion of approximately 83 amino acids from the amino-terminus of
STATs has been demonstrated to impair the ability of STATs to bind to
tandem, but not to single GAS binding sites.53 In addition,
a point mutation at the extreme amino-terminus of STAT1 has been
demonstrated to result in its constitutive tyrosine phosphorylation,54 suggesting that this region may be
important for the interaction with a negative regulator such as a
phosphatase. The point mutants STAT5A/WKR and STAT5A/RQQ are located in
highly conserved regions of unknown function just C-terminal to this area. STAT5A/RQQ mutation had no effect on STAT5 heterodimerization, DNA binding, or JAK-dependent transactivation and, as expected, did not
have a dominant negative effect on erythropoietin-induced STAT5-dependent transcriptional activation. Interestingly, a point mutation in an adjacent residue (H299R) that is not highly conserved, in conjunction with a second point mutation in the C-terminus, induces
constitutive tyrosine phosphorylation and DNA binding of the mutant
STAT5A.26 We did not observe any evidence of constitutive tyrosine phosphorylation or transactivation with any of our STAT5A mutants. In contrast, the nearby STAT5A/WKR mutation disrupted STAT5
heterodimerization, despite a lack of effect on tyrosine phosphorylation, suggesting that this region of the STAT5A may act to
stabilize dimerization. Consistent with its defect in dimerization, STAT5A/WKR completely lacked DNA binding to a FcRI-derived GAS probe
and demonstrated no significant transcriptional activation of a
-casein reporter. Interestingly, this mutant had a potent dominant
negative effect on erythropoietin-induced STAT5-dependent transcriptional activation, suggesting that STAT5A/WKR acts by blocking
wild-type STAT binding to the intracytoplasmic domain of the
erythropoietin receptor. This is also the likely mechanism used by a
previously described dominant negative STAT5A mutant, STAT5A/650,49 which is defective in tyrosine
phosphorylation, heterodimerization, DNA binding, and transcriptional activation.
Although STAT proteins lack classical DNA binding domains and
transactivation motifs, recent studies have provided considerable insight into regions important for STAT activation and DNA
binding.55-58 The STAT5A/EE and STAT5A/VVV mutants, located
in the putative STAT DNA-binding domain,57,58 were
generated because of the striking sequence homology among different
STATs family members within this region (Fig 1). Both of these
mutations completely eliminated FcRI GAS DNA-binding as assessed by
EMSA, despite preservation of tyrosine phosphorylation and
dimerization. These results are consistent with a previous study in
which the same mutations in STAT3 significantly impaired binding to an
M67-derived oligonucleotide.57 Interestingly, in the
context of STAT3, the VVVAAA mutant still had some detectable
DNA-binding activity, likely reflecting subtle differences in the
manner that different STAT proteins bind to distinct oligonucleotide
sequences. STAT5A/VVV was also unable to transcriptionally activate a
-casein luciferase reporter; however, STAT5A/EE, also lacking
FcRI-DNA-binding activity, still transactivated a -casein
reporter almost as well as wild-type STAT5A, showing that DNA-binding
as assessed by EMSA is not always predictive of transcriptional
activation in vivo. When assessed for possible dominant negative
activity, STAT5A/EE had little effect on the ability of erythropoietin
to activate STAT5-dependent transcription even when transiently
expressed at an eightfold excess, suggesting that its preserved
transactivation ability could compensate for its relative defect in DNA
binding. This finding differs from the behavior of an EEAA
mutation in STAT3, which does function as a dominant
negative.59 In contrast, STAT5A/VVV exerted a dominant
negative effect on erythropoietin-induced STAT5-dependent transcription
that increased with increasing levels of expression of the mutant STAT
protein. Because of its preserved heterodimerization ability,
STAT5A/VVV may exert its dominant negative effect by forming
heterodimers that are defective in DNA binding. It is also likely that
some of the dominant negative effect of STAT5A/VVV is due to
competition for binding sites on the cytoplasmic portion of the
erythropoietin and IL-3 receptors.
Previous studies of dominant negative STAT5 mutants have focused on
deletion mutagenesis of the carboxy-terminal portion of the protein.
Some STAT5 mutants have involved large carboxy-terminal deletions,
including the tyrosine (Y694) phosphorylated by JAK family
members,23,49 whereas others were constructed by deletion of a small C-terminal basic region implicated in transcriptional activation.24,25 We generated a similar mutant,
STAT5A/53C, by analogy to a naturally occurring isoform of rat
STAT5B,34 which also has dominant negative properties (H. Baumann, personal communication, January 1998). As
expected, the STAT5A/53 mutant exhibited JAK2-dependent tyrosine
phosphorylation, heterodimerization, and DNA binding, with an apparent
increase in the efficiency of DNA binding by EMSA, consistent with the
suggestion that the STAT5A C-terminus might also negatively regulate
DNA binding.24 Interestingly, we found that the 53C
truncation diminished, but did not completely eliminate,
transcriptional activation by the mutant STAT, suggesting that this
mutation has not completely abolished the transactivation function of
STAT5A. Despite this, the STAT5A/53C mutant functioned fairly
efficiently as a dominant negative, particularly when expressed at
higher levels. This suggests that, whereas some of the dominant negative action of STAT5A/53C is through the formation of
heterodimers with wild-type STAT5 that are defective in transcriptional
activation, the precise mechanism of interference with wild-type STAT5A
function is more complicated.
To determine the effects of inhibiting STAT5-dependent transcription in
hematopoietic cells, we expressed the STAT5A/WKR and STAT5A/53C
mutants in the IL-3-dependent myeloid cell line 32D. Attempts to
express these STAT5A mutants by low-efficiency transfection of cells
with plasmids capable of high level expression, such as pcDNA3, were
uniformly unsuccessful, whereas overexpression of wild-type STAT5A was
easily achieved (data not shown), suggesting that high-level expression
of these dominant negative STAT5 mutants was deleterious to the
proliferation or survival of these cells. We therefore turned to a
double-cistron retroviral vector with an internal ribosome entry site,
which permitted the efficient transduction and rapid selection of a
population of cells expressing both a neomycin resistance gene and the
dominant negative STAT. Using this system, we were able to obtain
populations of G418-resistant 32D cells expressing the dominant
negative STAT proteins at severalfold over the level of endogenous
STAT5A. When tested immediately after transduction and selection, we
observed significant inhibition of IL-3-induced STAT5-dependent
transcription (Fig 6) and proliferation (Fig 7) in cells expressing
either dominant negative STAT5 allele. These results confirm earlier
observations23,26 and suggest that a STAT5-dependent
pathway contributes to the proliferative response induced by IL-3 in
hematopoietic cell lines. Others have failed to observe an effect of a
similar C-terminal truncation mutant of STAT5 on proliferation upon
stable expression in 32D cells.25 The reason for the
discrepancy between these results and our observations are not clear,
but the cells in the prior study were selected for a cotransfected drug
resistance marker for 2 weeks followed by subcloning by limiting
dilution, lengthy procedures that may select for clones that can
compensate for the loss of STAT5 activity for proliferation. Although
STAT3 has been implicated in the generation of an antiapoptotic signal
in response to activation of the gp130 receptor,60 we and
others23,25 observed no increase in cell death in cells
expressing dominant negative STAT5 mutants, suggesting that STAT5 does
not play a major role in the IL-3-dependent antiapoptotic response in
these cells.
Interestingly, we found that expression of both our dominant negative
STAT5 mutants profoundly inhibited G-CSF-induced neutrophil differentiation of 32D cells, suggesting a novel role for STAT5 in
myeloid cell differentiation as well as proliferation. Because 32D
cells are an immortalized cell line, it is possible they do not
faithfully model the granulocytic differentiation process in bone
marrow. Importantly, we also observed a reproducible and significant
impairment of G-CSF-dependent granulocytic colony formation upon
expression of the dominant negative STAT5 mutants in primary murine
bone marrow, confirming a role for STAT5 activation in neutrophil
maturation. At first glance, these results appear to be in conflict
with the phenotype of stat5a/
stat5b/ double knockout mice, which
have apparently normal steady-state myelopoeisis. Unlike other gene
products, such as C/EBP ,61 STAT5 is clearly not
absolutely required for granulocyte development when it is lacking from
the moment of fertilization. However, we note that marrow from
stat5a/
stat5b/ double knockout mice exhibits
about a 50% reduction in G-CSF-dependent granulocytic colony
formation relative to wild-type marrow,21 which is also
consistent with a contribution of STAT5 to neutrophil development.
G-CSF receptor stimulation has been shown to activate JAK1 and JAK2 and
lead to tyrosine phosphorylation and activation of STAT3, STAT1, and
STAT5.13,14 Studies of G-CSF receptor (G-CSF-R) mutants
demonstrate that G-CSF-induced differentiation signals require the
distal (C-terminal) region of the intracytoplasmic domain of G-CSF-R,
distinct from the membrane-proximal region necessary for activation of
JAK kinases and for G-CSF-induced proliferative
effects.62,63 Whereas efficient activation of STAT3
requires the presence of at least one of four conserved tyrosine
residues present in the distal region of the G-CSF-R,13,64 activation of STAT5 by G-CSF appears to be independent of G-CSF-R phosphorylation.13 Expression of dominant-negative STAT3
mutants in myeloid cell lines blocked G-CSF-induced neutrophil
differentiation52 and IL-6-induced macrophage
differentiation,59 suggesting a functional role for STAT3
in cytokine-induced myeloid differentiation. Our results suggest that
STAT5 activation is also required for the G-CSF-induced neutrophil
maturation process. The involvement of STAT5 in both proliferative and
maturation responses is interesting, because differentiation is
intimately coupled with growth arrest in hematopoietic cells, so that
proliferation and maturation are to some degree intrinsically
antagonistic processes. However, it is very clear that both growth and
differentiation are complex processes involving multiple signaling
pathways, and activation of STAT5 is neither absolutely required for
proliferation13,23,25 nor sufficient to induce
differentiation.30,65 Complete understanding of myeloid
cell growth and maturation as well as myeloid leukemogenesis will
require the identification of STAT5-induced genes involved in these
processes, which should be facilitated by the use of the mutants we
have described here.
| |
ACKNOWLEDGMENT |
The authors thank Dr Alan D'Andrea for providing the STAT5A 650
mutant before its publication; Dr Don Wojchowski for the gift of the
dominant negative JAK2 mutant; Dr Mark Showers for the gift of the
erythropoietin receptor expression plasmid and for reading the
manuscript; Dr Bernd Groner for gift of the luciferase plasmids; Dr Jim
Griffin for gift of 32D Cl3 cells; Dr Alice Mui for the gift of probes
for CIS, PIM-1, and OSM; Vinay Kumar and P.V.
Sivakumar for assistance with FACS analysis; and Dr Heinz Baumann for
helpful advice.
| |
FOOTNOTES |
Submitted July 28, 1998; accepted February 16, 1999.
Supported by National Institutes of Health Grants No. HL03310 (R.L.I.)
and CA57593 (R.A.V.). R.A.V. is a Scholar of the Leukemia Society of
America and the Carl and Margaret Walter Scholar in Blood Research at
Harvard Medical School.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Richard A. Van Etten, MD, PhD, Center for
Blood Research, 200 Longwood Ave, Boston, MA 02115; e-mail:
vanetten{at}cbr.med.harvard.edu.
| |
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ABSTRACT
INTRODUCTION