The Journal of Experimental Medicine • Volume 192, Number 5, September 4, 2000 719–728
Essential Role of Signal Transducer and Activator
of Transcription (Stat)5a but Not Stat5b for
By Shuli Zhang,
and Hal E. Broxmeyer
R osanne Spolski,
Warren J. Leonard,
Oncology Center , Indiana University School of Medicine , Indianapolis , Indiana 46202; the
Cancer Institute , Indianapolis , Indiana 46206; and the
National Heart, Lung, and Blood Institute , Bethesda, Maryland 20892
Departm ent of Microbiology/Im m unology, the
Departm ent of Medicine , and the
Laboratory of Molecular Im m unology,
The receptor tyrosine kinase Flt3 plays an important role in proliferation and survival of he-
matopoietic stem and progenitor cells. Although some post-receptor signaling events of Flt3
have been characterized, the involvement of the Janus kinase/signal transducer and activator of
transcription (Jak/Stat) pathway in Flt3 signaling has not been thoroughly evaluated. To this
aim, we examined whether Flt3 activates the Jak/Stat pathway in Baf3/Flt3 cells, a line stably
expressing human Flt3 receptor. Stat5a, but not Stats 1–4, 5b, or 6, was potently activated by
Flt3 ligand (FL) stimulation. Interestingly, FL did not activate any Jaks. Activation of Stat5a re-
quired the kinase activity of Flt3. A selective role for Stat5a in the proliferative response of pri-
mary hematopoietic progenitor cells to FL was documented, as FL did not act on progenitors
from marrows of Stat5a
mice, but did stimulate/costimulate proliferation of these cells from
, Stat5b, and Stat5b mice. Thus, Stat5a is essential for at least certain effects of
FL. Moreover, our data confirm that Stat5a and Stat5b are not redundant, but rather are at least
partially distinctive in their function.
hematopoiesis • gene knockout
Flt3 • signal transducer and activator of transfection 5 • signal transduction •
Flt3 ligand (FL)
cally with a wide range of CSFs and ILs to stimulate prolif-
eration and differentiation of hematopoietic stem and pro-
genitor cells (1–4). It also enhances survival of progenitor
cells (5, 6). Its receptor, Flt3, belongs to the type III recep-
tor tyrosine kinases (R TKs) that also include receptors for
CSF-1, Steel (or stem cell) factor (SLF), and platelet-derived
growth factor (PDGF; reference 7). This family has five
immunoglobulin-like domains in their extracellular region
and an intracellular tyrosine kinase made up of an ATP-
binding loop and a catalytic domain separated by a kinase
is a potent cytokine that acts synergisti-
insert domain. In vivo and in vitro studies have shown that
FL and Flt3 play important roles in proliferation and differ-
entiation of both multipotent stem and lymphoid progeni-
tor cells (1–4, 8–10). Mice lacking Flt3 receptor have nor-
mal mature hematopoietic populations; however, they
exhibit reduced numbers of primitive B lymphoid progeni-
tors and multipotent stem cells (11).
Before the cloning of Flt3 ligand, the capacity of the Flt3
receptor to transmit a signal was studied by constructing
chimeric receptors between the cytoplasmic and trans-
membrane domains of Flt3 and the extracellular domains of
either the c-kit (12) or CSF-1 receptor (13–15). Using this
strategy, some early signaling events and substrate specifici-
ties of Flt3 receptor were characterized (13, 15, 16). The
chimeric receptor transduced mitogenic signals through as-
sociation with and/or phosphorylation of a number of cy-
toplasmic proteins, including R AS GTPase–activating pro-
tein, phospholipase C-
, Vav, Grb2, Shc, and the p85
subunit of phosphatidylinositol 3 kinase. p85 has been
Address correspondence to Hal E. Broxmeyer, Department of Microbiol-
ogy/Immunology and the Walther Oncology Center, Indiana University
School of Medicine, Bldg. R 4, R m. 302, 1044 West Walnut St., India-
napolis, IN 46202-5254. Phone: 317-274-7510; Fax: 317-274-7592;
Abbreviations used in this paper: FL, Flt3 ligand; Jak, Janus kinase;
PDGF, platelet-derived growth factor; R TK, receptor tyrosine kinase;
SLF, Steel factor; Stat, signal transducer and activator of kinase.
Flt3 Ligand Preferentially Activates STAT5A in a JAK-independent Manner
shown to bind tyrosine 958 (YQNM) in the COOH-ter-
minal tail of murine Flt3 (15, 16). R ecently, we found that
p85 does not bind directly to human Flt3, which lacks a
potential p85-binding site in the COOH terminus (17). In
addition, FL stimulation of the human Flt3 receptor re-
sulted in phosphorylation of Src homology 2 (SH2)-con-
taining inositol-5-phosphatase (SHIP) and a 100-kD pro-
tein in monocytic THP-1 cells (17); phosphorylation of
Shc and Cbl in myeloid cells (18); and SH2-containing ty-
rosine phosphatase, SHIP, and Cbl-b in pro-B cells (17, 19,
20). However, whether Janus kinase/signal transducer and
activator of transcription (Jak/Stat) proteins are involved in
Flt3 signaling has not been clearly studied.
In this study, we examined whether Flt3 activates the
Jak/Stat pathway in a murine IL-3–dependent hematopoi-
etic cell line Baf3, which stably expresses full-length human
Flt3 receptor. We found that, unlike most R TKs, Flt3 po-
tently activates only Stat5a without activation of any Jaks.
This activation of Stat5a by Flt3 was also observed in COS-7
cells cotransfected with Flt3 receptor and Stat5 expression
vectors. Moreover, FL did not stimulate/costimulate pro-
liferation of primary hematopoietic progenitor cells from
marrows of mice functionally deleted in Stat5a (
did act normally on Stat5a littermate control (
), and Stat5b (
) progenitor cells. These results
demonstrate that Stat5a, but not Stat5b, plays a critical role
in mediating FL effects.
) but it
Materials and Methods
Cytokines and Antibodies.
rine SLF were provided by Immunex. R ecombinant preparations
of murine IL-3, GM-CSF, M-CSF, and G-CSF were purchased
from R &D Systems. R abbit polyclonal anti-Flt3, anti-Stat5a,
anti-Stat5b, anti-Stat3, anti-Stat6, and anti-Tyk2 antibodies were
purchased from Santa Cruz Biotechnology, Inc. R abbit poly-
clonal anti-Stat5a, anti-Stat5b, anti-Jak1, anti-Jak2, and anti-Jak3
antibodies and antiphosphotyrosine mAb (4G10) were obtained
from Upstate Biotechnology. Anti-Stat1, anti-Stat2, and anti-
Stat4 antibodies were from Transduction Laboratories. AG490
and AG1296 were from Calbiochem-Novabiochem.
Cell Culture, DNA Constructs, and Transfection.
IL-3–dependent hematopoietic cell line Baf3/Flt3, a subline of
Baf3 transduced with the human Flt3 receptor gene, was cultured
as previously described (17). Before cytokine stimulation, Baf3/
Flt3 cells were washed with PBS and starved in serum-free R PMI
1640 overnight. COS-7 and HEK293 cells were cultured in
DMEM containing 10% fetal bovine serum and 100 U/ml peni-
cillin and streptomycin.
The cDNA encoding human Flt3 (provided by Dr. Steward
Lyman, Immunex, Seattle, WA) was subcloned into the expres-
sion vector pCDNA3 (Invitrogen). Mutants of Flt3 that lack ei-
ther COOH terminus (Flt3-
CT; amino acids 950–993) or both
COOH terminus and the second kinase domain (Flt3-
TKII; amino acids 782–993) were constructed by PCR -based
mutagenesis. The cDNAs encoding murine Stat5a and Stat5b
were obtained from Dr. X .-Y. Fu (Yale University, New Haven,
CT). Transfection of COS-7 and 293 cells was performed using
SuperFect reagent (Qiagen) according to the manufacturer’s pro-
tocol. For cytokine stimulation experiments, COS-7 or 293 cells
R ecombinant human FL and mu-
were serum-starved for 24 h and then stimulated with FL (100
Im m unoprecipitation and Im m unoblotting.
were suspended in serum-free media (1–2
stimulated at 37
C with human FL or murine IL-3 in the absence
of any serum. Cell extracts were prepared and subjected to im-
munoprecipitation and immunoblotting with the antibodies indi-
cated as previously described (20).
Electrophoretic Mobility Shift Assay.
treated and FL-treated cells were prepared as described elsewhere
(21). For the EMSA, 20,000 cpm of
quence motif corresponding to the
g of nuclear extract proteins in 20
ing 10 mM Tris, pH 7.5, 50 mM NaCl, 1 mM dithiothreitol, 1
mM EDTA, 0.05% NP-40, 2
10% glycerol at room temperature for 30 min and then resolved
on 4% polyacrylamide gels containing 0.5
mmol/liter Tris borate and 1 mmol/liter EDTA, pH 8) and 2.5%
glycerol. Gels were run at 4
C in 0.5
and autoradiographed. Oligonucleotide competition was per-
formed by preincubating nuclear extracts with the cold probe
(50-fold excess) and poly(dI·dC):poly(dI·dC) for 30 min at 4
before the addition of labeled probe. For supershifts, samples
were preincubated for 1 h at 4
C with the indicated antibodies.
Mice.Mice functionally deleted (
Stat5b (23) were from fifth generation backcrosses to the C57BL/6
background. These mice were generated from heterozygous mat-
ings. Some of the characteristics of immune cells from these mice
have been described previously (24, 25). As controls, cells from
age- and sex-matched littermate Stat5a
Hem atopoietic Progenitor Cell Assay.
10 cells/ml) obtained from femurs of Stat5a
, Stat5b, and Stat5b
(Difco) culture medium in the presence of 10% heat-inactivated
fetal bovine serum (Hyclone) in the absence and presence of
varying concentrations of human FL; murine SLF; murine GM-
CSF; murine M-CSF; human G-CSF; the combination of SLF
with GM-CSF, M-CSF, or G-CSF; or the combination of FL
with SLF, GM-CSF, M-CSF, or G-CSF. Plates were incubated
at 5% CO
and lowered (5%) O
sphere and scored for colonies deriving from granulocyte and/or
macrophage progenitor cells (26). Three plates were scored per
point per experiment.
Flow Cytom etric Analysis.After lysis of red blood cells, mouse
bone marrow cells were subjected to negative selection using lin-
eage-specific antibodies (anti-Gr1, anti-CD11b, anti-CD3, and
anti-B220 antibodies from PharMingen) and complement to re-
move granulocytes, macrophages, and mature T and B cells.
Then the cells were stained with fluorochrome-conjugated anti-
bodies (anti–Flt3–PE and anti–c-kit–FITC from PharMingen)
and analyzed using FACScan
with CELLQuest software (Bec-
After starvation, cells
10 cells/ml) and
Nuclear extracts from un-
-casein gene promoter (5
) was incubated with 20
l of binding buffer contain-
g poly(dI·dC):poly(dI·dC), and
TBE is 89
TBE at 20 V/cm, dried,
) in Stat5a (22) and
Unseparated bone mar-
mice were plated in 0.3% agar
for 7 d in a humidified atmo-
FL Preferentially Activates Stat5a over Stat5b in Baf3/Flt3
To test whether any Stat proteins are involved in
Flt3 signaling, we used Baf3/Flt3 cells transduced with full-
length Flt3 cDNA. This subline stably expresses Flt3 recep-
tor on the cell surface and proliferates in response to human
Zhang et al.
FL (20). Among the Stat proteins we examined (Stat1, 2, 3,
4, 5, and 6), only Stat5 was tyrosine phosphorylated by FL
stimulation, while IL-3 induced tyrosine phosphorylation
of both Stat3 and Stat5 (Fig. 1 A). Tyrosine phosphoryla-
tion of Stat5 induced by FL was transient and reached a
maximal level at 5 min (Fig. 1 B). It was weak compared
with IL-3 stimulation. Fig. 1 C shows the dose–response
of tyrosine phosphorylation of Flt3 and Stat5 induced by
FL stimulation. We also checked Stat1, 2, 4, and 6. Al-
though they are expressed in Baf3/Flt3 cells, none of them
were tyrosine phosphorylated by FL stimulation (data not
shown). These results demonstrate that unlike most R TKs,
Flt3 selectively activates Stat5.
Two different Stat5 genes, encoding Stat5a and Stat5b
90% identity at the amino acid level, have
been identified (27–30). Both Stat5a and Stat5b are acti-
vated by many cytokines and growth factors. The targeted
knockout of the individual genes in mice has suggested that
they play essential and at least partially overlapping roles in
the physiological responses associated with several cyto-
kines (22–25, 31). Despite their homology, there is evidence
suggesting that Stat5a and Stat5b may be differentially acti-
vated (32). Since the anti-Stat5 antibody we used in Fig. 1
recognizes both Stat5a and Stat5b, we assessed whether FL
activated both or only one of the Stat5 proteins. By using
specific anti-Stat5a and -Stat5b antibodies, we found that
FL induced tyrosine phosphorylation of Stat5a but not
Stat5b (Fig. 2 A). IL-3 induced tyrosine phosphorylation of
both Stat5a and Stat5b in Baf3/Flt3 cells (data not shown).
We next investigated if FL could stimulate Stat5a DNA
binding activity by EMSAs using a
motif from the
-casein gene promoter, which is known to
bind to both Stat5a and Stat5b. As shown in Fig. 2 B, FL
stimulation induced a DNA binding complex that was
clearly supershifted by anti-Stat5a antibody, but anti-Stat5b
antibody had a much smaller effect. This is consistent with
the tyrosine phosphorylation pattern of Stat5 and suggests
that gel shift assays tend to be more sensitive than phospho-
tyrosine blots. These results demonstrate that FL preferen-
tially activates Stat5a in Baf3/Flt3 cells.
FL Does Not Activate Jaks.
FL stimulation, we tested whether Jaks are activated by FL.
Jak1, Jak2, Jak3, and Tyk2 were immunoprecipitated from
cells treated with either FL or IL-3, separated by SDS-
PAGE, transferred to polyvinylidene difluoride mem-
branes, and blotted with antiphosphotyrosine antibody. IL-3
has been reported to activate both Jak1 and Jak2 (33–35).
As shown in Fig. 3, although IL-3 induced rapid tyrosine
phosphorylation of Jak1 and Jak2, none of the Jaks were
activated by FL. These results show that FL does not acti-
vate Jaks and suggests that activation of Stat5a is likely me-
diated by other tyrosine kinases, possibly the Flt3 receptor.
Since Stat5a is activated by
starved Baf3/Flt3 cells were stimulated with FL (100 ng/ml) or IL-3 (10 ng/ml) for 5 min. Stat5
and Stat3 were immunoprecipitated from cell lysates and immunoblotted with antiphosphoty-
FL induces tyrosine phosphorylation of Stat5 in Baf3/Flt3 cells. (A) Growth factor-
rosine antibody. (B) Stat5 is transiently tyrosine phosphorylated by FL stimulation. Growth factor–starved Baf3/Flt3 cells were stimulated with FL for
various periods of time or with IL-3 for 5 min. Stat5 was immunoprecipitated from cell lysates and immunoblotted with antiphosphotyrosine antibody.
(C) Dose–response of tyrosine phosphorylation of Flt3 and Stat5. Growth factor–starved Baf3/Flt3 cells were stimulated with different dose of FL for 5
min. Flt3 and Stat5 were immunoprecipitated from cell lysates and immunoblotted with antiphosphotyrosine antibody. The same membranes were
stripped and reblotted with different antibodies as shown. R esults shown are from one representative of two to three experiments. pY, antiphosphoty-
Flt3 Ligand Preferentially Activates STAT5A in a JAK-independent Manner
Activation of Stat5a Requires the Kinase Activity of Flt3.
Since Stat5a is tyrosine phosphorylated after FL stimulation
and none of the Jaks is activated by FL stimulation, it is pos-
sible that Flt3 may directly phosphorylate Stat5a. To test
this hypothesis, we first used type III receptor tyrosine ki-
nase inhibitor AG1296, which has been shown to specifi-
cally inhibit SLF and PDGF signaling (36). As a control, we
used AG490, which inhibits the kinase activity of Jak2 and
Jak3 (37, 38). As shown in Fig. 4 A, AG1296 at 50
minished tyrosine phosphorylation of Flt3 to the control
level, whereas AG490 did not have an effect. After Baf3/
Flt3 cells were pretreated with these two inhibitors, Stat5
was immunoprecipitated from cells treated with FL and ty-
rosine phosphorylation of Stat5 was determined by Western
blot analysis with antiphosphotyrosine antibody. As shown
in Fig. 4 B, tyrosine phosphorylation of Stat5a was de-
creased close to control levels by AG1296, but not by
AG490. This is consistent with the results that Jaks were not
activated by FL stimulation and suggests that the tyrosine
kinase activity of Flt3 is required for activating Stat5a.
Next, we transiently transfected Flt3 cDNA with either
Stat5a or Stat5b cDNA into COS-7 and HEK293 cells and
examined whether Flt3 could induce tyrosine phosphoryla-
tion and DNA binding activity of Stat5. After serum-star-
vation for 24 h, the transfected cells were stimulated with
human FL (100 ng/ml) for 5 min. Stat5a and Stat5b were
immunoprecipitated from cell lysates and tyrosine phos-
phorylation was analyzed by immunoblotting with an-
tiphosphotyrosine antibody. As shown in Fig. 5 A, no
phosphorylation of either Stat5a or Stat5b was detected in
COS-7 cells that were mock transfected. Cotransfection of
Flt3 and Stat5a cDNA resulted in strong phosphorylation
of Stat5a. Addition of FL slightly increased phosphorylation
of Stat5a. In contrast, the phosphorylation of Stat5b was
very weak, and could only be detected after longer expo-
sure when coexpressed with Flt3. Control immunoblots
showed that equivalent levels of Flt3 were expressed. Simi-
lar results were obtained when using 293 cells (data not
shown). We then evaluated the DNA binding activity of
Stat5 by EMSAs. As shown in Fig. 5 B, no DNA binding
activities were detected in COS-7 cells that were trans-
fected with vector control plus Stat5a or Stat5b alone.
Cotransfection of Flt3 cDNA with Stat5a resulted in high
levels of a DNA complex that was supershifted by anti-
Stat5a antibody. Although Stat5a and Stat5b were ex-
pressed at similar levels (data not shown), cotransfection of
Flt3 with Stat5b yielded only a weaker DNA binding activ-
tivates Stat5a in Baf3/Flt3 cells.
(A) Growth factor–starved Baf3/
Flt3 cells were stimulated with
FL for 5 min. Stat5a and Stat5b
were immunoprecipitated from
cell lysates and immunoblotted with anti-phosphotyrosine antibody (pY).
The same membrane was stripped and reblotted with anti-Stat5 antibody
that recognizes both forms of Stat5. R esults shown are from one repre-
sentative of three experiments. (B) FL activates Stat5a DNA binding ac-
tivity. Growth factor–starved Baf3/Flt3 cells were either untreated (con-
trol) or stimulated with FL for 10 min. Nuclear extracts from these cells
were incubated with 32P-labeled probe and subjected to EMSA. Oligonu-
cleotide competition was performed by preincubating nuclear extracts
with the cold probe. For supershifts, nuclear extracts were preincubated
with the indicated anti-Stat5a or anti-Stat5b antibodies. The arrow indi-
cates the DNA/Stat5 complex. R esults shown are from one representa-
tive of two experiments.
FL preferentially ac-
stimulation in Baf3/Flt3 cells. Growth fac-
tor–starved Baf3/Flt3 cells were stimulated
with FL (100 ng/ml) or IL-3 (10 ng/ml) for
5 min. Jak1, Jak2, Jak3, and Tyk2 were im-
munoprecipitated from cell lysates and im-
munoblotted with antiphosphotyrosine an-
tibody (pY). The same membranes were
stripped and reblotted with different anti-
bodies as shown in the figure. R esults
shown are from one representative of three
Jaks are not activated by FL
Zhang et al.
ity. These results confirmed that Flt3 preferentially acti-
Although expression of wild-type Flt3 in COS-7 cells
led to phosphorylation of Stat5a, mutant Flt3 lacking the
second kinase domain did not induce phosphorylation of
Stat5a, and mutant Flt3 lacking the COOH terminus in-
duced less phosphorylation of Stat5a (Fig. 5 C). Wild-type
and mutants of Flt3 were expressed at similar levels as mea-
sured by flow cytometry (data not shown). These results
demonstrate that phosphorylation of Stat5a requires ty-
rosine kinase activity of Flt3. Thus, Stat5a is either a direct
substrate of Flt3, or Flt3 activates an endogenous tyrosine
kinase that in turn phosphorylates Stat5a, or both.
Myeloid Progenitors from Stat5a
the Stim ulating/Costim ulating Effe c ts of FL.
mice functionally deleted in genes for certain intracellular
molecules has allowed the elucidation of the roles of these
gene-encoded proteins in blood cell regulation (39). To
this end, we evaluated the responsiveness in vitro of mye-
loid progenitor cells from femurs of Stat5a
mice to the proliferation effects of FL. By itself, FL is a
weak stimulator of the proliferation of myeloid progenitor
cells. However, in combination with GM-CSF, M-CSF,
or G-CSF, it is a potent costimulating molecule that acts
synergistically with these CSFs to enhance the number and
size of colonies (4).
Mice Do Not Respond to
Use of cells from
tion of Flt3 and Stat5a by AG1296, but not by
AG490. Baf3/Flt3 cells were pretreated with ei-
ther control diluent DMSO or AG1296 and
AG490 for 1 h at 37?C and washed once with
PBS. Then the cells were treated with FL for 5
min. Flt3 (A) and Stat5a (B) were immunopre-
cipitated from cell lysates and immunoblotted
with antiphosphotyrosine antibody (pY). The
same membranes were stripped and reblotted
with different antibodies as shown in the figure.
In B, AG1296 and AG490 were used at 50 ?M.
R esults shown are from one representative of
Inhibition of tyrosine phosphoryla-
COS7 cells. (A) COS-7 cells were transfected with
Stat5a, Stat5b, Flt3, or control vector in the combi-
nations shown. After stimulation with either me-
dium control or FL, cells were lysed and Stat5a or
Stat5b was immunoprecipitated from cell lysates and
immunoblotted with antiphosphotyrosine antibody.
The same membrane was stripped and reblotted
with anti-Stat5 antibody that recognizes both Stat5a and Stat5b. The expression of Flt3 was confirmed by immunoblot with anti-Flt3 antibody on total cell
lysates. (B) Nuclear extracts from COS7 cells transfected with Stat5a, Stat5b, Flt3, or vector control in the combination shown were incubated with 32P-labeled
probe and subjected to EMSA. Oligonucleotide competition was performed by preincubating nuclear extracts with the cold probe. For supershifts, nuclear
extracts were preincubated with the anti-Stat5a or -Stat5b antibodies. The arrow indicates the DNA–Stat5 complex. (C) COS-7 cells were transfected with
Stat5a plus control vector, Flt3, Flt3-?CT, or Flt3-?CT/TKII, as shown. The tyrosine phosphorylation of Stat5a was examined by immunoprecipitation
and immunoblotting with antiphosphotyrosine antibody. The same membrane was stripped and reblotted with anti-Stat5a antibody. pY, antiphosphotyro-
sine antibody; SS, supershift.
Flt3 preferentially activates Stat5a in
Flt3 Ligand Preferentially Activates STAT5A in a JAK-independent Manner
Bone marrow cells from Stat5a
mice were plated in the absence and presence of
varying concentrations of FL, SLF, GM-CSF, M-CSF, or
G-CSF, or varying concentrations of SLF or FL with each
other, or varying concentrations of each of the CSFs (Fig.
6). FL by itself had minimal activity on stimulation of col-
ony formation of Stat5a
progenitors and little or no
stimulation of Stat5a
progenitors. No significant differ-
0.05) were noted in response of Stat5a
progenitors to stimulation by SLF, GM-CSF,
M-CSF, or G-CSF, or to the synergistic effects of 50 ng/ml
SLF plus GM-CSF, M-CSF, or G-CSF. However, the co-
stimulating synergistic effects of 100 ng/ml FL with SLF,
GM-CSF, M-CSF, or G-CSF seen with cells from Stat5a
0.001) were not apparent with cells from
mice. The effects of FL in combination with the
different concentrations of SLF or CSFs on Stat5a
were equivalent to the single actions of these concentrations
of SLF or CSFs. Similar differences were noted using 10
and littermate control
ng/ml FL or SLF with varying concentrations of CSFs (data
not shown). Greater than 90% of colonies stimulated with
M-CSF with or without FL or SLF were composed purely
of macrophages. Greater than 90% of colonies stimulated
with G-CSF with or without FL or SLF were composed of
neutrophilic granulocytes. Colonies stimulated with GM-
CSF with or without FL or SLF were
granulocytes and macrophages with the remainder being
pure granulocyte or pure macrophage colonies.
To evaluate the specificity of these effects, similar com-
parative experiments were done with myeloid progenitor
cells from Stat5b
and their littermate control Stat5b
mice (Fig. 7). No significant differences (
noted in the responses of Stat5b
the individual effects of FL, SLF, GM-CSF, M-CSF, G-CSF,
or to the combined effects of SLF or FL with each other
or with any of the CSFs. At the same time these experi-
ments were done, experiments were also done with Stat5a
and Stat5a cells with results comparable to those seen in
These results demonstrate the critical role played by
Stat5a, but not Stat5b, in myeloid progenitor cell respon-
50% composed of
tor cells from marrows of Stat5a??? but not Stat5a??? mice. R esults are
shown as the average percentage of control of cells incubated with 50 ng/
ml SLF plus 10 ng/ml GM-CSF for a total of six experiments each. The
control colony numbers upon which the percent of control are based
were 52 ? 2 (mean ? 1 SEM), 70 ? 3, 89 ? 4, 60 ? 4, 152 ? 3, and
132 ? 2 for Stat5a??? cells and 62 ? 3, 87 ? 3, 101 ? 8, 71 ? 2, 70 ?
2, and 55 ? 3 for Stat5a??? cells.
FL stimulates/costimulates proliferation of myeloid progeni-
tor cells from marrows of Stat5b??? and Stat5b??? mice to the same ex-
tent. R esults are shown as the average percentage of control of cells incu-
bated with 50 ng/ml SLF plus 10 ng/ml GM-CSF for a total of two
experiments each. The control colony numbers upon which the percent-
age of control are based were 66 ? 2 and 93 ? 1 for Stat5b??? cells, and
93 ? 2 and 74 ? 3 for Stat5b??? cells.
FL stimulates/costimulates proliferation of myeloid progeni-
Zhang et al.
siveness to the stimulating/costimulating effects of FL. To
make sure that the differences noted between Stat5a???
cells compared with those of Stat5a???, Stat5b???, and
Stat5b??? cells were not due to differential expression of
Flt3 on the cells, protein levels of Flt3 were assessed by
flow cytometry on the surface of bone marrow cells that
were depleted of lineage-positive cells. (Fig. 8). Expression
of Flt3 on Stat5a??? c-kit?Lin? cells, a population highly
enriched for myeloid progenitor cells (40), was at least as
high as on the Stat5a???, Stat5b???, and Stat5b??? cells.
Thus, the expression levels of Flt3 on the cells from these
mice could not explain the nonresponsiveness of Stat5a???
progenitors to stimulation/costimulation by FL.
In this study we have found that FL preferentially acti-
vates Stat5a over Stat5b, and that myeloid progenitors from
the bone marrow of Stat5a???, Stat5b???, and Stat5b???,
but not from Stat5a???, mice respond to the stimulating/
costimulating effects of FL. These results demonstrate that
Stat5a plays a critical role in mediating FL effects. More-
over, this adds additional evidence to the literature showing
the nonoverlapping effects of Stat5a and Stat5b.
Among the different Stat proteins examined, only Stat5a
was potently tyrosine phosphorylated by FL stimulation. FL
did not seem to activate any Janus kinase as measured by
phosphotyrosine immunoblotting (Fig. 3). Longer stimula-
tion with FL (30 min) did not result in any phosphorylation
of Jak1 and Jak2 (data not shown). This is in contrast to
most growth factors whose receptors are tyrosine kinases,
such as PDGF, SLF, and M-CSF (or CSF-1). PDGF, SLF,
and M-CSF have been shown to activate multiple Jak and
Stat proteins. M-CSF induces tyrosine phosphorylation of
Jak1, Tyk2, and Stat1, 3, and 5; PDGF activates Jak1, Jak2,
Jak3, and Stat1, 3, 5, and 6 (41–44); and SLF activates Jak2
and Stat1, 3, and 5 (45, 46). These growth factors activate
both Stat5a and Stat5b. Our results suggest that FL differs
from these R TKs in that it activates only Stat5a without an
apparent activation of Jaks.
R ecently, a group reported that a tandem-duplicated
form of Flt3 constitutively activates Stat5 and mitogen-acti-
vated protein (MAP) kinase, whereas FL stimulation leads
to activation of MAP kinase but not Stat5 activation in
Baf3 and 32D cells expressing wild-type Flt3 (47). In that
study (47), it was not specified whether Hayakawa et al.
were dealing with Stat5a or Stat5b or both Stat5 proteins.
Our data show that FL stimulation of wild-type Flt3 pref-
erentially activates Stat5a, and our in vivo data strongly
suggest that Stat5a, but not Stat5b, plays a critical and po-
tentially physiological role in mediating the synergistic ef-
fects of FL. Although the same cell line, Baf3, was used, the
discrepancy between the study by Hayakawa et al. and ours
may be due to different levels of Flt3 expression and the as-
Stat5a and Stat5b are two highly related transcription
factors encoded by two different genes. Stat5a and Stat5b
are activated by a broad range of cytokines and growth fac-
tors. Although most cytokines and growth factors activate
both Stat5a and Stat5b, there are some studies showing that
Stat5a and Stat5b can be differentially activated and regu-
late different sets of genes. For example, GM-CSF prefer-
entially activates Stat5b, but not Stat5a, in human neutro-
phils, whereas IFN-? and -? predominantly activate Stat5a
in promonocytic U937 cells (48, 49). Insulin preferentially
activates Stat5b, but not Stat5a, via a Jak2-independent sig-
naling pathway in kym-1 rhabdomyosarcoma cells (50).
Not only can Stat5a and Stat5b be differentially activated,
they also have distinctive functions. In support of this,
Stat5a??? and Stat5b??? mice have different phenotypes
(22–25, 51). Our findings that FL preferentially activates
Stat5a in both Baf3/Flt3 and COS7 cells and that myeloid
progenitors from the marrow of Stat5a???, Stat5b???, and
Stat5b???, but not from Stat5a???, mice respond to the
stimulating/costimulating effects of FL provide another ex-
ample of differential activation of Stat5 protein. Moreover,
our results strongly suggest that Stat5a, but not Stat5b, plays
a critical role in myeloid progenitor cell responsiveness to
the stimulating/costimulating effects of FL.
The Jak/Stat pathway has been well characterized in cy-
tokine signaling. In the type I cytokine receptor family,
ligand stimulation induces rapid activation of Jaks which
then phosphorylate Stat proteins. In contrast, in tyrosine
kinase receptors, the role of Jak proteins in Stat activation is
not clearly defined. Some R TKs like PDGFR and epider-
mal growth factor receptor (EGFR ), although they can ac-
tivate Jaks, activate Stat proteins in a Jak-independent fash-
ion (43, 52). In contrast, the activity of the intrinsic R TK is
absolutely required for Stat activation by EGF or PDGF. A
mutant PDGFR , which lacks kinase activity, is unable to
of Stat5a???, Stat5a???, Stat5b???, and Stat5b??? mice as analyzed by flow
cytometry. Mouse bone marrow cells were depleted of lineage-positive
cells and stained with anti–Flt3–PE and anti–c-kit–FITC antibodies.
Large granulocytes and debris were gated out on the basis of their light
scatter pattern. The quadrants were set up based on isotype control anti-
bodies. The numbers shown on the two-dimensional dot plots are per-
centages of single positive cells (Flt3? or c-kit?) and double positive cells
Flt3 receptor expression on the surface of bone marrow cells
Flt3 Ligand Preferentially Activates STAT5A in a JAK-independent Manner
stimulate Stat1 and Stat3 activation in response to PDGF
stimulation (43). Stat1 can directly interact with EGF,
PDGF, and SLF receptors and these receptors can phos-
phorylate Stat1 in vitro (46, 53, 54). EGFR can also di-
rectly phosphorylate Stat3 in vitro (55). These results sug-
gest that Stat proteins may be direct substrates of receptor
tyrosine kinases. Our findings that the kinase activity of
Flt3 is required for Stat5a activation supports this hypothe-
sis. However, our results do not rule out the possibility that
Flt3 may activate an endogenous tyrosine kinase that in
turn phosphorylates Stat5a. R egardless of the mechanism,
our data confirm that Stat5a and Stat5b are not redundant,
but rather are at least partially distinctive in their functions.
We would like to thank Dr. Stewart Lyman for Baf3 and Baf3/Flt3
cells; Dr. X.-Y. Fu (for Stat5a and Stat5b cDNA; and Dr. Mark Kap-
lan and Charlie Mantel for reading the manuscript and for their
This work was supported by US Public Health Service grants
R 01 HL56416 and R 01 DK53674 to H.E. Broxmeyer.
Subm itted: 1 June 2000
Revised: 10 July 2000
Accepted: 17 July 2000
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