From the
Interleukin-3 (IL-3)-, erythropoietin (EPO)-, and prolactin
(PRL)-induced signal transduction via the JAK/STAT pathway was studied
in the IL-3-dependent BAF-3 lymphoid cell line. Transfected cells
expressing either the long form of the PRL receptor or the EPO receptor
were used. We demonstrated that IL-3, EPO, and PRL activated a
transcription factor related to the mammary transcription factor STAT5
but not to STAT1, -2, -3, or -4 as opposed to interferon
Signal transduction of interferons (IFNs)
PRL also plays an immunomodulatory
role, lymphocytes being its main target(4) . PRL receptors
(PRL-R) belong to the cytokine receptor superfamily (5) which
includes multiple receptors expressed in hematopoietic cells such as
receptors for interleukin (IL-) 2, 3, 4, 5, 6, 7, 9, 11, and 12,
erythropoietin (EPO), thrombopoietin, and others (6). PRL signal
transduction begins to be elucidated in a limited number of
hematopoietic cells expressing either endogenous PRL-R (Nb2 rat T
lymphoma cell line) or transfected PRL-R (the BAF-3 IL-3-dependent
lymphoid cell line and the FDCP1 IL-3-dependent myeloid cell
line)(7, 8, 9, 10) . In these cells, PRL
activates the rapid and transient tyrosine phosphorylation of various
proteins including its receptor and kinases of the JAK family, either
JAK2 in Nb2, or JAK2 and weakly JAK1 in
BAF-3(9, 11, 12, 13) . Furthermore, in
the Nb2 lymphoma cells, PRL induces the expression of an immediate
early gene, the interferon (IFN) regulatory factor 1
(IRF1)(7, 8) , which regulates the expression of both
IFNs and IFN-induced genes(14, 15) . Recent studies on
IRF1 indicated that its promoter contains one critical GAS element
suggesting that the PRL-induced IRF1 expression could be induced by a
JAK/STAT pathway in hematopoietic
cells(1, 16, 17) .
Two recent studies
indicated that in Nb2 lymphoma cells, PRL activated two members of the
STAT family, one related to STAT1 and another unknown protein of 97
kDa(12, 18) . Because of the expression of STAT5 in the
spleen and thymus, we first wondered whether STAT5 could be activated
by PRL in hematopoietic cells. Moreover, since the mammary STAT5 was
shown to be a substrate of kinase JAK2(3) , we investigated
whether cytokines known to activate JAK2 such as IL-3 or
EPO(19, 20) also triggered the activation of STAT5.
The BAF-3 cells express endogenous IL-3 receptors and are strictly
dependent on the presence of IL-3 for growth. Transfection with either
the mammary PRL-R or the EPO-R confers to these cells the ability to
grow in the presence of PRL or EPO, respectively. Here we describe that
in transfected BAF-3 cells, PRL, IL-3, and EPO activate a
tyrosine-phosphorylated DNA binding factor, of 97 kDa, highly related
to STAT5 but not to STAT1, -2, -3, or -4, as opposed to IFN
The
anti-STAT antibodies used were: anti-STAT1 N-terminal domain
(anti-ISGF3 p91/84 monoclonal antibody, Transduction Laboratories, UK),
anti-STAT2 peptide (gift from C. Schindler(30) ), anti-STAT3
C-terminal peptide (30C, gift from D. Levy, New York University Medical
Center (21)), anti-STAT4 C-terminal peptide (C-20, Santa Cruz
Biotechnology, Inc.), anti-STAT5 N-terminal fusion protein containing
amino acids 6 to 160 of sheep mammary STAT5(31) , and an
anti-STAT raised against a STAT3 N-terminal peptide which recognizes
all STAT members (21) (30N gift from D. Levy, named thereafter
PANSTAT). Antiphosphotyrosine antibody 4G10 was kindly provided by Dr.
B. Drucker (Portland).
For supershift assays, an excess
of antibodies was added to the nuclear extracts just prior to the
addition of the probe.
Nuclear extracts from BAF-3 cells transfected with the PRL-R (9) and treated with PRL were prepared. Band shift assays were
carried out with a labeled probe from the
We next asked whether the activation of
STAT5-L was restricted to PRL. Like the parental BAF-3 cells, BAF-3
cells transfected with the PRL-R were responsive to IL-3(9) .
Also, BAF-3 cells expressing a transfected murine EPO-R that
proliferated in the presence of EPO had been established. Nuclear
extracts from either IL-3-treated PRL-R-transfected or EPO-treated
EPO-R-transfected cells were analyzed by EMSA using either the IRF1
probe or
We thank Dr. D. Levy for the generous gift of
anti-STAT3 and anti-PANSTAT antibodies and Dr. C. Schindler for
anti-STAT2 antibodies. We also thank Dr. S. Pellegrini for her critical
reading of the manuscript.
Note Added in Proof-Two
murine STAT
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(IFN
) which activated STAT1 in the same cells. Similarly, PRL and
EPO activated a STAT5-like factor (STAT5-L) in the rat Nb2 and the
human UT7 cells expressing endogenous PRL and EPO receptors,
respectively. The hematopoietic STAT5-L activated by IL-3, EPO, or PRL
was identified as a 97-kDa tyrosine-phosphorylated protein. These
results confer to STAT5 a much broader role than previously suggested.
(
)has served as a model system for studies on
activation of the JAK kinases and downstream-located latent cytoplasmic
transcriptional factors known as STATs (for review see Refs. 1 and 2).
Six members of the STAT family are presently known. These factors are
characterized by their rapid activation through tyrosine
phosphorylation(1) . STAT1 and STAT2, both activated by
IFN
, associate with a 48-kDa DNA-binding protein and translocate
to the nucleus where they bind interferon-stimulated response element
(ISRE), while only STAT1 is activated by IFN
and binds
-activated sequences (GAS). STAT5-MGF was first isolated as a
mammary transcriptional factor (MGF) specifically activated by the
hormone prolactin (PRL) in mammary epithelial cells, inducing the
expression of milk proteins(3) . However, the expression of
STAT5 was not restricted to the mammary gland and was also detected in
hematopoietic organs(3) .
which
activates STAT1. This STAT5-like factor is also activated by PRL or EPO
in hematopoietic cells expressing endogenous PRL- or EPO-R,
respectively.
Hormones and Antibodies
Ovine prolactin
(NIDDK6-PRL-19, 31 IU/mg) was a gift from the National Hormone and
Pituitary Program (Baltimore, MD). Recombinant EPO, IFN, and IL-3
were from Boehringer and Pepro Tech. Inc., respectively.
Cell Culture
BAF-3 cells were transfected with
cDNA coding for either the long form of the rabbit mammary PRL-R or the
murine EPO-R, both cloned in the pBabeNeo expression vector, as
described(9) . BAF-3 cells were cultured in RPMI 1640 medium
supplemented with 7% fetal calf serum (Dutscher) and either 5% culture
supernatants from the IL-3-producing Wehi-3B cell line (Wehi-CM) or 10
ng/ml PRL or 2 units/ml EPO(9) . Nb2-11C cells (22) were
maintained in RPMI 1640 medium supplemented with 7% fetal calf serum.
UT7 cells (23) were cultured in -medium supplemented with
10% fetal calf serum and 2.5 ng/ml granulocyte macrophage-colony
stimulating factor (5
10
units/mg) as
described(24) .
Nuclear Extracts
Cells were first deprived of
cytokines and of fetal calf serum by incubation for 4 h (for BAF-3 and
Nb2 cells) or 18 h (for UT7 cells) in Iscove's medium
supplemented with 0.4% detoxified bovine serum albumin (Fraction V,
Sigma) and 75 µg/ml iron-saturated transferrin (Sigma) (9) and then incubated in the presence of either 1-1000
ng/ml PRL or 10 ng/ml IL-3, 10 units/ml EPO, or 1000 units/ml IFN
for the indicated time. Nuclear extracts were prepared by first lysing
the cells in a hypotonic buffer (20 mM Hepes, pH 7.9, 10
mM KCl, 1 mM EDTA, 0.2% Nonidet P-40, 10% glycerol)
supplemented with 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml pepstatin A, 10
µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM DTT and
extracting the pelleted nuclei in a hypertonic buffer (0.35 M NaCl, 20% glycerol, 20 mM Hepes, pH 7.9, 10 mM KCl, 1 mM EDTA, 1 mM DTT, with the same protease
and phosphatase inhibitors being added). After 30 min at 4 °C,
extracts were centrifuged for 5 min at 4 °C at 20,000
g, and supernatants were immediately frozen in liquid nitrogen
and stored at -80 °C.
Electrophoretic Mobility Shift Assay
(EMSA)
Nuclear extracts (10
cells/point) were
incubated with 16 fmol of
P-labeled oligonucleotide
containing STAT5 binding site from either the bovine
-casein
promoter (5`-AGATTTCTAGGAATTCAAATC-3`) or the GAS element of the IRF1
promoter (5`-GATCCATTTCCCCGAAATGA-3`). These oligonucleotides were
end-labeled with polynucleotide kinase to a specific activity of 8000
cpm/fmol as described(3) .
Purification of the DNA Binding Activity
Nuclear
extracts (10 cells) in the binding buffer (10 mM Tris, pH 7.5, 1 mM EDTA, 1 mM DTT, 0.1% Nonidet
P-40, 5% glycerol) containing 100 mM NaCl were incubated with
Sepharose beads coupled to multimerized STAT5 binding sites from the
-casein gene promoter (3) for 45 min at 4 °C in the
presence of 20 µg of poly(dI-dC) and 20 µg of poly(dA-dT).
After washing, proteins bound to the Sepharose beads were eluted with
the SDS loading buffer, separated by SDS-polyacrylamide gel
electrophoresis (8%), and transferred to nitrocellulose membrane
(Schleicher and Schuell, BA85). Blots were incubated first with a STAT5
antibody (1/1000), then stripped and further incubated with the
phosphotyrosine antibody (1/5000).
-casein promoter
containing the STAT5 binding site. As shown in Fig. 1A (lanes 1-5), PRL induced the activation of a DNA
binding complex that appeared in a very rapid and transient manner,
becoming almost undetectable after 60 min of incubation. This DNA
binding activity was induced in the presence of concentrations of PRL
(10 ng/ml) required for proliferation of these BAF-3 cells (Fig. 1A, lanes 9-12)(9) . To
assess further the induction of a STAT5-related DNA binding activity,
EMSAs were extended to another STAT5-DNA binding sequence, the GAS of
the IRF1 promoter. This motif contains a palindromic motif
5`-TTCXXXGAA-3` which is strictly required for STAT5 DNA
binding(25, 31) . As shown in Fig. 1A, lanes 6-8, PRL-treated cells possessed a DNA binding
activity with the GAS-IRF1 oligonucleotide. This activity was induced
as early as 1 min after PRL addition (Fig. 1B, lanes
1-4). Similar results were obtained with the
-casein
probe.
Figure 1:
PRL
activates a STAT5 DNA binding activity in BAF-3 and in Nb2 cells. A and B, nuclear extracts from BAF-3 cells expressing PRL-R
were stimulated with various concentrations of PRL (ng/ml) for the
indicated times (min). Nuclear extracts were analyzed in EMSAs using
either -casein or IRF1 oligonucleotide as probes. C,
nuclear extracts from PRL-R-transfected BAF-3 cells stimulated for 20
min with 1 µg/ml PRL were incubated with various antisera against
either STAT1, STAT2, STAT3, STAT4, or STAT5 (S
, S
, S
, S
, S
) or PANSTAT (PS) and with the
-casein probe. Shifted and supershifted complexes (SS)
are pointed out. D, nuclear extracts from Nb2 cells (lanes
1-7) or PRL-R-transfected BAF-3 cells (lanes 8 and 9) treated for O or 10 min with 1 µg/ml PRL were incubated
with the indicated antisera against either STAT1, -2, -3, -4, or -5 (S
, S
, S
, S
, S
)
and with the IRF1 probe. EMSAs were performed. Shifted and supershifted (SS) complexes are pointed out.
The identity of the activated STAT factor was investigated by
incubating nuclear extracts from PRL-stimulated cells with various
STAT-specific antisera and analyzing them by EMSA (Fig. 1C). DNA binding complexes with the IRF1 probe
were supershifted only with STAT5 (lane 6) and PANSTAT (lane 7) antisera. Neither STAT1 nor STAT2, -3, or -4 antisera
were able to recognize the complexes, indicating that STAT5 or a
STAT5-like (STAT5-L) factor, was specifically activated by PRL in the
PRL-R-transfected BAF-3 cells. To strengthen this result, we prepared
nuclear extracts from another murine lymphoid cell line, the Nb2 cells,
which express large amounts of endogenous PRL-R(26) . As shown
in Fig. 1D, PRL induced the rapid and transient
activation of a DNA binding complex (lane 2). Supershift
experiments (lanes 3-7) indicated that this complex
contained both a STAT5-L protein (lane 7) and STAT1 (lane
3). It is worth noting that STAT1 activation was only detectable
at high PRL concentrations.
-casein probe. As shown in Fig. 2A, IL-3 (lanes 4-6) and EPO (lanes 7-9) induced
binding activity to the
-casein probe. Complexes formed after PRL
stimulation (Fig. 2A, lanes 1-3) migrated
the same as the IL-3- or EPO-stimulated complexes. The PRL-, IL-3-, or
EPO-induced complexes were all supershifted with STAT5 antiserum, but
not with antisera directed against STAT1, STAT2, STAT3, or STAT4 (Fig. 2B). We next checked the activation of STAT5-L
factors by EPO in the human UT7 cell line which expressed high amounts
of endogenous EPO-R(27) . As shown in Fig. 2C, in
the UT7 cell line, EPO activated a transcriptional factor which
migrated the same as the one induced by EPO in BAF-3 cells (lane
1). Supershift experiments confirmed that this factor is highly
related to STAT5 (lane 8) and not to STAT1, -2, -3, or -4.
Figure 2:
A STAT5 DNA binding activity is activated
by EPO and IL-3. A, BAF-3 cells expressing either PRL-R (lanes 1-6) or EPO-R (lanes 7-9) were
stimulated with 1 µg/ml PRL, 10 ng/ml IL-3, or 10 units/ml EPO.
Nuclear extracts were analyzed by EMSA with the -casein probe. B, nuclear extracts from IL-3- (10 ng/ml, 10 min) or EPO- (10
units/ml, 10 min) stimulated BAF-3 cells expressing the EPO-R were
incubated with various antisera against either STAT1, STAT2, STAT3,
STAT4, or STAT5 (S
, S
, S
, S
, S
)
and with the IRF1 probe. EMSAs were performed. Shifted and supershifted (SS) complexes are pointed out. C, nuclear extracts
from UT7 cells (lanes 3-8) or EPO-R-transfected BAF-3
cells (lanes 1 and 2) treated with 10 units/ml EPO
for O or 10 min were prepared and analyzed by
EMSA.
BAF-3 cells also expressed endogenous IFN-R. EMSA of
IFN
-treated cells indicated that IFN
rapidly and transiently
induced a DNA binding complex with the IRF1 probe (Fig. 3A, lane 2, and Fig. 3B, lanes 1-5) or
-casein probe (not shown) which
migrated faster than the complex induced by IL-3 (Fig. 3A, lanes 1 and 2)) or PRL (not
shown). Supershift assays indicated that this IFN
complex
contained STAT1 but not STAT3 or -5 (Fig. 3B, lanes
6-10). STAT5 was not activated by IL-6 in the U937 monocytic
cell line, although in those cells, IL-6 induced the activation of
STAT3 which bound the IRF1 probe and which was recognized by the
anti-STAT3(31) . These data showed that STAT5-L proteins are
specifically involved in PRL, EPO, and IL-3 signaling.
Figure 3:
IFN activates STAT1 in
BAF-3 cells. PRL-R-transfected BAF-3 cells were incubated with IL-3 (10
ng/ml, A, lane 1) or IFN
(1000 units/ml, A, lane 2, and B) for the indicated time.
Nuclear extracts were analyzed by EMSA with the IRF1 probe. Supershift
assays were carried out with nuclear extracts from cells treated for 20
min with IFN
in the presence of the indicated anti-STAT
antibodies: anti-STAT1, -3, -5 (S
, S
, S
) and PANSTAT antibody (PS). Supershifted complexes are indicated (SS).
To better
characterize STAT5-L proteins, nuclear extracts from PRL-, IL-3-, or
EPO-treated or from untreated BAF-3 cells were incubated with Sepharose
beads coupled to a multimerized STAT5 binding site from the
-casein promoter. Bound proteins were analyzed by
SDS-polyacrylamide gel electrophoresis followed by immunoblotting first
with a STAT5 antiserum and then with an antiphosphotyrosine antibody.
Both antibodies recognized one band of 97 kDa, in PRL-, IL-3-, or
EPO-activated extracts (Fig. 4).
Figure 4:
Identification of the STAT5-L DNA binding
activity induced by PRL or IL-3 or EPO. EPO-R-transfected BAF-3 cells (lanes 1 and 2) or PRL-R-transfected BAF-3 cells (lanes 3-5) were incubated without (lanes 1 and 3) or with 10 units/ml EPO (lane 2), 1 µg/ml PRL (lane 4), or 10 ng/ml IL-3 (lane 5) for 20 min.
Nuclear extracts were incubated with multimerized -casein
oligonucleotides coupled to Sepharose beads. Bound proteins were
analyzed by immunoblotting with STAT5 specific antiserum (top)
and then reprobed with antiphosphotyrosine antibody (bottom).
Our data indicate that in a
variety of hematopoietic cells expressing endogenous or transfected
receptors (murine BAF-3, rat Nb2, and human UT7) PRL, IL-3, and EPO
rapidly activate a transcriptional factor immunologically closely
related to STAT5, but not to STAT1, -2, -3, or -4. Moreover, this
factor becomes tyrosine-phosphorylated following ligand action. Whether
this hematopoietic factor is totally identical with the mammary STAT5
protein recently isolated or corresponds to a new highly related STAT
is under investigation. It is worth noting that preliminary data(
)suggest the existence of two genes for STAT5. Whether
one encodes the mammary STAT5 form and the other the hematopoietic
STAT5 is currently unknown. Our findings are in agreement with a number
of reports on the activation of unknown STATs by either EPO,
granulocyte macrophage-colony stimulating factor, or PRL in
hematopoietic
cells(8, 12, 13, 21, 28, 29) .
In agreement with our data, these unidentified STATs may be the
hematopoietic STAT5 protein, suggesting a wide activation of STAT5 in
hematopoietic cells. Such results pointed out a general role of STAT5
in cell activation. One main target should be the tumor suppressor IRF1
gene whose expression was reported to be induced by a wide variety of
cytokines in various cells, including BAF-3 cells (7, 8, 14, 15).
Alternatively, it could indicate that STAT5 participates in a variety
of specific transcriptional complexes.
-activated
sequence; IRF1, interferon regulatory factor 1; DTT, dithiothreitol;
EMSA, electrophoretic mobility shift assay.
homologs have now been cloned (32, 33) and designated STAT
and
STAT
.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.