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Molecular and Cellular Biology, September 1999, p. 6396-6407, Vol. 19, No. 9
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Suppression of STAT5 Functions in Liver, Mammary
Glands, and T Cells in Cytokine-Inducible SH2-Containing Protein 1 Transgenic Mice
Akira
Matsumoto,1
Youichi
Seki,2
Masato
Kubo,2
Satoshi
Ohtsuka,1
Asuka
Suzuki,1
Itsuro
Hayashi,3
Kohichiro
Tsuji,4
Tatsutoshi
Nakahata,4
Masaru
Okabe,5
Shuichi
Yamada,5 and
Akihiko
Yoshimura1,*
Institute of Life Science, Kurume University,
Kurume 839-0861,1 Division of
Immunobiology, Research Institute for Biological Sciences, Science
University of Tokyo, Noda 278-0022,2
Department of Pathology (II), Faculty of Medicine, Kurume
University, Kurume 830-0011,3 Department
of Clinical Oncology, The Institute of Medical Science, The
University of Tokyo, Minatoku, Tokyo 108-8639,4
and Genome Information Research Center, Osaka University,
Suita 565-0871,5 Japan
Received 16 February 1999/Returned for modification 8 April
1999/Accepted 22 June 1999
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ABSTRACT |
Various cytokines utilize Janus kinase (JAK) and the STAT (signal
transducers and activators of transcription) family of transcription factors to carry out their biological functions. Among STATs, two
highly related proteins, STAT5a and STAT5b, are activated by various
cytokines, including prolactin, growth hormone, erythropoietin, interleukin 2 (IL-2), and IL-3. We have cloned a STAT5-dependent immediate-early cytokine-responsive gene, CIS1 (encoding
cytokine-inducible SH2-containing protein 1). In this study, we created
CIS1 transgenic mice under the control of a 
-actin promoter. The
transgenic mice developed normally; however, their body weight was
lower than that of the wild-type mice, suggesting a defect in growth
hormone signaling. Female transgenic mice failed to lactate after
parturition because of a failure in terminal differentiation of the
mammary glands, suggesting a defect in prolactin signaling. The
IL-2-dependent upregulation of the IL-2 receptor 
chain and
proliferation were partially suppressed in the T cells of transgenic
mice. These phenotypes remarkably resembled those found in STAT5a
and/or STAT5b knockout mice. Indeed, STAT5 tyrosine phosphorylation was
suppressed in mammary glands and the liver. Furthermore, the
IL-2-induced activation of STAT5 was markedly inhibited in T cells in
transgenic mice, while leukemia inhibitory factor-induced STAT3
phosphorylation was not affected. We also found that the numbers of
T cells, as well as those of natural killer (NK) cells and NKT
cells, were dramatically decreased and that Th1/Th2 differentiation was
altered in transgenic mice. These data suggest that CIS1 functions as a
specific negative regulator of STAT5 in vivo and plays an important regulatory role in the liver, mammary glands, and T cells.
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INTRODUCTION |
The STAT (signal transducers and
activators of transcription) proteins are cytosolic latent
transcription factors that are rapidly activated by interferons (IFNs),
cytokines, and growth factors (4, 9, 26). A total of seven
different STAT proteins, many of which play highly specific roles in
innate and acquired immunity, have been identified. Mice with STAT1,
STAT4, and STAT6 gene knockouts are viable but lack functions that are
mediated by IFNs, interleukin 12 (IL-12), or IL-4/IL-13, respectively
(5, 13, 20, 29, 33). In contrast, STAT3 knockout mice
exhibit fetal lethality (34), a finding consistent with the
activation of STAT3 by many cytokines that are important for
development, such as leukemia inhibitory factor (LIF), cardiotrophin-1,
and IL-6 (4, 9). The highly related proteins STAT5a and
STAT5b are activated by various cytokines, including growth hormone
(GH), prolactin (PRL), erythropoietin (EPO), IL-2, IL-3,
granulocyte-macrophage colony-stimulating factor (GM-CSF), and
thrombopoietin. The phenotypes of STAT5a and STAT5b single-gene
knockout mice and double-knockout mice revealed important roles of
STAT5 in PRL and GH signaling as well as in IL-2-dependent T-cell
functions (7, 11, 17, 23, 35, 36). STAT5a is critical in PRL
signaling (17), GM-CSF signaling (7), or IL-2
signaling (23), while STAT5b is necessary to maintain the
sexual dimorphism of body growth rates and liver gene expression
(36), IL-2-mediated T-cell proliferation, and NK cell
development (11, 21).
The regulation of the Janus kinases (JAKs) and STATs is a central
component in the control of cytokine signaling. Because of the critical
role of cytokines in mediating inflammation and immunity, it could be
proposed that the constitutive activation of JAKs could contribute to
hematopoietic disorders, autoimmunity, and inflammatory diseases.
However, the mechanisms that terminate or down-modulate the JAK-STAT
pathway have not been fully understood. Considerable evidence suggests
that one mechanism involves the recruitment of a tyrosine phosphatase
containing the SH2 domain (SHP-1) to receptor complexes, resulting in
the dephosphorylation of JAKs (12, 14). The potential
importance of this mechanism is strongly suggested by the phenotype of
motheaten (me/me) mice lacking SHP-1, which die from a disease with
components of autoimmunity and inflammation (30). However,
it has also been suggested that a family of small SH2 domain-containing
proteins may be involved in the relatively specific regulation of
cytokine signaling.
The CIS (cytokine-inducible SH2-containing protein) family of proteins,
also referred to as the SOCS (suppressors of cytokine signaling) or SSI
(STAT-induced STAT inhibitor) family, has been implicated in regulating
signal transduction by a variety of cytokines (1, 31, 43).
The gene encoding first member of this family, CIS1, was cloned as an
immediate-early gene responding to a number of cytokines, including
EPO, IL-2, IL-3, and GM-CSF (41), and is regulated by STAT5
(19). CIS1 binds to the tyrosine-phosphorylated IL-3 and EPO
receptors and negatively regulates their signals (41). The
second family member was independently cloned by three groups and is
termed JAB (JAK-binding protein), SOCS-1, or SSI-1 (6, 22,
31). JAB is induced by IFN- and inhibits IFN as well as IL-6
signaling (28, 32). JAB and CIS3 directly bind to the kinase
domain of JAKs, thereby inhibiting the kinase activity (32).
This family now contains eight members, although most of them have not
been well characterized except for CIS1, CIS3, and JAB (18,
43).
The CIS1 promoter contains two pairs of tandem TTCNNNGAA motifs
that are capable of binding to STAT5 (19). The essential role of STAT5 in CIS1 expression was confirmed by the observation that
CIS1 expression was not observed in the ovaries of STAT5a and -b
double-gene knockout mice (35). In addition to
STAT5-dependent expression of CIS1, it was interesting that CIS1 could
negatively modulate STAT5 activation (19); forced expression
of CIS1 partially suppressed EPO-dependent STAT5 activation in 293 and
Ba/F3 cells. Thus, we hypothesized that CIS1 acts as a kind of negative
feedback regulator of the JAK-STAT5 pathway.
To elucidate the physiological function of CIS1 and to find the
relationship of the dysregulated expression of the CIS1 gene to human
diseases, we created CIS1 transgenic mice. A striking similarity of
phenotypes was found between CIS1 transgenic mice and STAT5 knockout
mice. The CIS1 transgenic mice exhibited growth retardation and defects
in mammary gland development as well as in T-cell and NK cell
development. In addition, we found that helper T cells of CIS1
transgenic mice tend to differentiate into Th2 cells, suggesting an
important role of CIS1 in T-cell differentiation. We also showed a
decrease in STAT5 activation in response to GH, PRL, and IL-2 in
transgenic mice. This study suggests an important regulatory role of
CIS1 and STAT5 in T-cell development and differentiation, in addition
to PRL and GH functions.
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MATERIALS AND METHODS |
Generation of CIS1 transgenic mice.
The pCAGGS-CIS1
expression vector was constructed by subcloning the myc-tagged CIS1
cDNA (18) into the EcoRI cloning site of the
pCAGGS (24). After digestion with SalI and
HindIII, the fragment carrying the promoter, cDNA, and
the 3' noncoding region was used for microinjection into (C57BL/6 × C3H)F1 (B6C3-F1) zygotes. Eggs surviving microinjection
were transferred into the oviducts of recipient pseudopregnant females
as described elsewhere (10). Transgenic mice were identified
by PCR analysis of tail genomic DNA, and protein expression was
confirmed by immunoblotting with anti-Myc.
Histology.
Virgin and lactating mice were sacrificed, and
mammary-gland tissue was taken, fixed in 10% formalin, paraffin
embedded, and sectioned. Mammary sections were stained with hematoxylin
and eosin and used for histological analysis.
Immunoblot analysis.
The postpartum mammary glands and other
tissues were immediately frozen in liquid nitrogen and stored at
80°C. Total-cell extracts were prepared from tissue homogenized in
50 mM Tris-HCl (pH 8.0)-0.5% Nonidet P-40-1 mM EDTA-150 mM
NaCl-10% glycerol-1 mM sodium vanadate-50 mM sodium fluoride-10 mM
sodium pyrophosphate-1 mM phenylmethylsulfonyl fluoride with protease
inhibitor cocktail (Sigma Chemical Co., St. Louis, Mo.). The extracts
were cleared by spinning at 15,000 rpm at 4°C for 15 min. The samples
were resolved by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE), and proteins were detected by
immunoblotting as described elsewhere (19). Anti-STAT5
(C-17) and anti-STAT3 (C-20) were purchased from Santa Cruz
Biotechnology, Inc. Tyrosine-phosphorylated STAT3 and STAT5 were
detected by using phosphospecific anti-STAT3 (New England Biolabs) and
anti-STAT5 (anti-pY-STAT3 and anti-pY-STAT5) (Upstate Biotechnology) as
described elsewhere (32). To detect the mobility shift of
STAT5, relatively small amounts of cell extracts (1 µg of protein for
mammary glands or 5 µg for the liver) were resolved by SDS-6% PAGE.
Northern hybridization.
Total RNA from various tissues was
prepared with TRIZOL (Gibco-BRL) according to the manufacturer's
instructions. Total RNA (5 µg) was separated on 1.0% agarose-2.4%
formaldehyde gels, then hybridized with digoxigenin-labeled riboprobes
prepared by using a digoxigenin-RNA labeling kit (Boehringer Mannheim
Japan, Tokyo) as described elsewhere (8, 32, 41).
Flow cytometry analysis.
Cells were suspended in
phosphate-buffered saline (PBS) supplemented with 2% fetal calf serum
and 0.1% sodium azide. In general, 106 cells were blocked
with anti-Fc receptor (2.4 G2) and stained by a standard method
described previously (15). Flow cytometry analysis was
performed with FACSort (Becton Dickinson, San Jose, Calif.) by using
CELL Quest software. The reagents (fluorescein isothiocyanate
[FITC]-conjugated anti-CD4 [RM4-5], anti-CD3 [125-2C11], anti-B220 [RA3-6B2], anti-T-cell receptor [anti-TCR] [H57-597], and anti-IL-2 receptor chain (7D4); phycoerythrin [PE]-conjugated anti-CD8 [53-6.7], anti-immunoglobulin M [anti-IgM], anti-NK1.1 [PK136], and anti- TCR [GL3]) were purchased from Pharmingen (La Jolla, Calif.).
Preparation of splenic T cells and IL-2-induced proliferative
responses.
To remove B cells and monocytes, spleen cells were
cultured in plates precoated with anti-mouse IgG for 1 h.
Unattached cells were used as splenic T cells. The splenic T cells were
preactivated with a plate-bound anti-TCR monoclonal antibody (MAb)
(H57-597) in the presence or absence of an anti-CD28 MAb (PV-1) (1 µg/ml). To isolate CD4+ naive T cells, spleen cells were
incubated with an anti-CD8 MAb (53-6.72) at 4°C for 1 h, and the
cells were incubated on plates coated with anti-mouse Ig to eliminate B
and CD8+ T cells. The CD4+ enriched T cells
were incubated with an anti-CD44 MAb (1M7), followed by a cytotoxic
killing treatment with Low-Tox-M rabbit complement (Cedarlane
Laboratories Limited, Hornby, Ontario, Canada). These CD4+
naive T-cell preparations contained more than 80% CD4+
CD44
T cells. The CD4+ naive T cells were
preactivated with a plate-bound anti-TCR MAb. After 36 h, the T
cells were cultured with various concentrations of IL-2 either in the
presence or in the absence of phorbol myristate acetate (PMA) (50 ng/ml) for 48 h and then pulse-labeled with 1 µCi of
[3H]thymidine for an additional 8 h. Recombinant
mouse IL-2 was purchased from PeproTech EC Ltd. (London, England).
Induction of helper T cells and intracellular cytokine
staining.
The CD4+ naive T cells were stimulated with
an anti-TCR MAb plus an anti-CD28 MAb in the presence or absence of
IL-4 as described previously (2). After 5 days, the primed
CD4+ T cells were repurified by panning with an anti-CD8
MAb and anti-mouse Ig and were restimulated with an anti-TCR MAb for
6 h in the presence of 4 µM monensin (Sigma). Then the cells
were fixed with 4% paraformaldehyde and permeabilized with 0.5%
Triton X-100. After the cells were blocked with PBS containing 3%
bovine serum albumin, cells were stained with anti-IFN-
(XMG1.2)-FITC and anti-IL-4 (11B11)-PE as described previously
(39). All antibodies were purchased from Pharmingen.
Detection of MUPs.
Major urinary proteins (MUPs) were
detected by SDS-15% PAGE and staining with Coomassie blue in 0.25 µl of urine as described previously (36).
Luciferase assay.
The STAT5-responsive promoter-luciferase
reporter gene, which carries four repeats of the IFN- activation
site (GAS) sequence with the jun promoter, was described
previously (18). GH- and PRL-dependent luciferase activity
in 293 cells grown in six-well plates was measured after transfection
with the reporter gene (50 ng of plasmid), the -galactosidase gene
(50 ng), murine GH or PRL receptor cDNA (200 ng), the indicated amount
of Flag-tagged CIS1 cDNA, and murine STAT5a or STAT5b (5 to 10 ng) as
previously described (18, 32, 40). For GH stimulation, serum
was omitted from the medium because of the high level of GH in serum.
Murine GH receptor cDNA cloned by PCR was subcloned into pcDNA3. Murine PRL receptor cDNA in pECE was donated by Wolfgang Doppler (Innsbruck University, Innsbruck, Austria). Murine STAT5a and -b cDNAs in pMX were
provided by Toshio Kitamura (Tokyo University, Tokyo, Japan).
Recombinant ovine GH and PRL were kind gifts from Arieh Gertler (The
Hebrew University of Jerusalem).
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RESULTS |
Growth retardation and defects of GH-induced STAT5 activation in
CIS1 transgenic mice.
CIS1 transgenic mice were generated by the
injection of Myc-epitope-tagged CIS1 cDNA with chicken -actin
promoter into B6C3-F1 eggs. Myc-CIS1 expression was examined by
Northern blotting and immunoblotting with either anti-Myc or anti-CIS1
antibodies, and three transgenic lines were established. One of the
lines (Tg1) expressed the transgenic CIS1 gene in the mammary glands
but not much in the liver and spleen (Fig.
1A; see also Fig. 4A and 6A). The other
two lines (Tg2 and Tg3) expressed transgenes in all tissues examined
(thymus, spleen, kidney, mammary glands, and liver), although Myc-CIS1
expression levels in most tissues was higher in Tg3 than in Tg2 (Fig.
1A, see also Fig. 6A). Tg2 and Tg3 lines, which express Myc-CIS1 in the
liver, showed a reduction in body weight compared with wild-type mice,
while the body weight of the Tg1 line, which did not express detectable
Myc-CIS1 in the liver, was normal (Fig. 1B). Growth retardation was
obvious in the Tg3 line from early ages, as shown in Fig. 1C, while Tg2 mice exhibited a normal growth rate until the age of 2 to 3 months but
did not gain much weight thereafter (Fig. 1B and data not shown). At
the age of 12 weeks, both the male and female Tg3 mice weighed 20 to
30% less than their wild-type littermates. The reduction in body
weight was more drastic in male than in female mice. Tg2 and Tg3 mice
had significantly less adipose tissue than wild-type mice (data not
shown). These phenotypes suggest a defect in GH signaling in CIS1
transgenic mice.
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FIG. 1.
Expression of Myc-CIS1 in the liver and body growth
rates of wild-type (WT) and CIS1 transgenic mice. (A) Exogenous
Myc-CIS1 expression in each transgenic line was detected by
immunoblotting of the liver extracts (10 µg of protein/lane) with
anti-Myc antibody (a). To ensure equal loading of the protein samples,
STAT5 was detected by an anti-STAT5 antibody (b). (B) Mean body weight
of each male transgenic line at the age of 9 months (n = 4). (C) Growth curves of WT and CIS1 transgenic male and female
mice from the age of 3 weeks through 12 weeks. The number of mice
analyzed (n) is shown. Standard errors of differences are
indicated on the x axis.
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To further examine the effect of CIS1 overexpression on GH signaling in
the liver, we compared the amount of MUP in the urine of wild-type and
transgenic mice. MUP is a family of 2-microglobulin-related liver
secretory proteins whose expression is regulated by male-specific pulsatile GH (25). As illustrated in Fig.
2A, the levels of MUP in both male and
female Tg3 mice (lanes 4 and 8) were much lower than those in wild-type
mice (lanes 1 and 5). The MUP levels of Tg2 CIS1 transgenic mice (lanes
3 and 7) were also lower than those in wild-type mice, but the
reduction was less drastic than that in Tg3 mice. We did not see any
reduction in MUP levels in Tg1 mice, which marginally expressed
transgenic CIS1 in the liver (Fig. 2A, lanes 2 and 6). These results
indicate that exogenous CIS1 expression levels correlate significantly
with the inhibition of GH-mediated gene expression in the liver.
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FIG. 2.
SDS-PAGE analysis of MUP in the urine (A) and activation
of STAT5 in the livers (B) of wild-type (WT) and CIS1 transgenic mice.
(A) Urine (0.25 µl) from male and female mice was resolved by
SDS-15% PAGE and stained with Coomassie blue. (B) Extracts (5 [a]
or 20 [b] µg of protein/lane) of freshly isolated livers from male
and female WT and CIS1 transgenic mice (lanes 1 to 4) were resolved by
SDS-6% PAGE and immunoblotted with an anti-STAT5 antibody (a) or
anti-pY-STAT5 (b). In lanes 5 and 6, purified bovine GH (4.8 mg/kg of
body weight) was injected intraperitoneally into female mice, and after
45 min, the livers were isolated. The slower electrophoretic form of
STAT5 is indicated as STAT5* in panel a. In panel b, the asterisk
indicates a nonspecific band observed in all lanes.
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GH exerts its function through the activation of STAT5. Since STAT5b
single-knockout mice as well as STAT5a,b double-knockout
mice also
exhibited similar defects in growth retardation and
MUP secretion
(
35,
36), we examined the effect of STAT5 activation
in the
livers of Tg3 mice, using immunoblotting with a phosphorylated
STAT5-specific antibody (Fig. 2Bb) and with the antibody that
recognizes both STAT5a and STAT5b (Fig. 2Ba). As shown in Fig.
2B,
STAT5 phosphorylation constitutively occurred in the livers
of
wild-type male mice (lane 1) and was detected by the mobility
shift of
the STAT5 band (Fig. 2Ba) with high-resolution SDS-PAGE
(
3,
27), as well as by immunoblotting with anti-phospho-STAT5
specific antibody (Fig. 2Bb). However, activation of STAT5 was
not seen
in CIS1 transgenic male mice (Fig.
2B, lane 2). STAT5
was not
constitutively activated in the livers of either wild-type
or
transgenic female mice (Fig.
2B, lanes 3 and 4). However, injection
of
GH intraperitoneally into female mice resulted in strong
phosphorylation
of STAT5 (Fig.
2B, lane 5). GH-induced phosphorylation
of STAT5
in the livers of transgenic female mice was less evident than
that in wild-type female mice (Fig.
2B, lane 6). This suggests
that
forced expression of CIS1 suppressed GH-dependent STAT5 activation
in
the
liver.
Defects in mammary-gland development and STAT5 activation in CIS1
transgenic mice.
All three transgenic lines expressed Myc-CIS1 in
mammary glands (Fig. 4A), and all of them exhibited defects in
mammary-gland development (Fig. 3),
although the fertility, length of gravidity, and litter size were
normal. All CIS1 transgenic females failed to lactate after
parturition, and milk was not detected in the stomachs of the pups
despite vigorous suckling by the pups. Upon fostering with wild-type
females, these pups thrived, which suggests that CIS1 transgenic
females could not produce or eject milk. Whole-mount analysis of
mammary tissue from postpartum CIS1 transgenic mice demonstrated
incomplete mammopoiesis (data not shown). Fig. 3 shows sections of
mammary gland tissues of virgin mice (A and E) and of mice 1 to 3 days
postpartum (B through D and F through H). The secretory tissue from
transgenic mice was sparse, and alveoli were petite and contained small
lumina (representative data for Tg3 mice are shown in Fig. 3).
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FIG. 3.
Histology of mammary glands from Tg3 mice. Mammary-gland
tissue sections (5 µm thick) from wild-type (WT) and transgenic
(CIS1-Tg) mice were stained with hematoxylin and eosin. Mature virgin
female mice at the age of 12 to 14 weeks (A and E) and 12- to
18-week-old female mice at 1 day, 2 days, or 3 days after delivery were
examined. Similar results were obtained for Tg1 and Tg2 mice.
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To examine STAT5 activation in mammary glands, tyrosine phosphorylation
of STAT5 was detected in tissue extracts postpartum.
The expression
levels of STAT5 in transgenic mice were slightly
lower than those in
wild-type mice (Fig. 4Ba). This may be
due
to a block of mammary-gland maturation during pregnancy, since
the
STAT5 level has been shown to increase at late pregnancy
(
16).
STAT5 bands were seen as doublets in wild-type mice,
probably
due to phosphorylation, but not in transgenic mice (Fig. 4Ba).
Tyrosine phosphorylation of STAT5 was very low in transgenic mice
(Fig.
4Bb). These data suggest that PRL-dependent STAT5 activation
is
inhibited in CIS1 transgenic mice.
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FIG. 4.
Expression of Myc-CIS1 (A), phosphorylation of STAT5
(B), and gene expression (C) in the mammary glands. (A) Exogenous
Myc-CIS1 expression in each transgenic line was detected by
immunoblotting of the mammary-gland extracts (10 µg/lane) from mature
virgin female mice with an anti-Myc antibody (a). To ensure equal
loading of the protein samples, STAT5 was detected with an anti-STAT5
antibody (b). WT, wild type. (B) Mammary-tissue extracts (1 [a] or 20 [b] µg/lane) from WT (lanes 1 to 3) or Tg3 (CIS1-Tg3; lanes 4 to 6)
mice at 1 day (lanes 1 and 4), 2 days (lanes 2 and 5), or 3 days (lanes
3 and 6) after parturition were immunoblotted with anti-STAT5 (a) or
anti-pY-STAT5 (b). STAT5*, slower electrophoretic form of STAT5. (C)
Northern blot analysis of total RNA (5 µg/lane) from mammary glands
of female mice at 1 day (lanes 1 and 4), 2 days (lanes 2 and 5), or 3 days (lanes 3 and 6) postpartum. The membrane was probed with
riboprobes for the genes for CIS1 (a), CIS2 (b), CIS3 (c), the milk
proteins -lactalbumin (d) and WAP (e), c-Myc (f), c-Fos (g), and
glyceraldehyde-3-phosphate dehydrogenase (G3PDH) (h). * and **,
endogenous and exogenous CIS1 mRNA, respectively.
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Expression of the endogenous CIS1 gene itself is strictly regulated by
STAT5 function (
7,
19,
21,
35). Therefore,
we measured CIS1
mRNA levels in mammary glands. As shown in Fig.
4Ca, exogenous CIS1
mRNA levels in Tg3 female mice were 3 to 5
times higher than endogenous
CIS1 mRNA levels in mammary glands
from pregnant female mice.
Endogenous CIS1 expression was suppressed
in transgenic mice compared
with that in wild-type mice, suggesting
low STAT5 activity in mammary
glands in transgenic mice. We also
examined the expression of other CIS
family genes because most
of them are cytokine inducible
(
18). CIS2 and CIS3 expression
were also detected, and the
levels were slightly increased during
lactation in wild-type mice (Fig.
4Cb and c). Interestingly, CIS2
and CIS3 expression, like that of
endogenous CIS1, was suppressed
in transgenic mice, although CIS3
reached normal levels at 3 days
postpartum. These data raise the
possibility that CIS2 and CIS3
are also regulated by
STAT5.
We next examined the level of milk protein, as well as c-Fos and c-Myc
levels, in mammary glands (Fig. 4Cd through h). CIS1
overexpression in
mammary glands resulted in a significant decrease
in whey acidic
protein (WAP) (e), while
-lactalbumin,
-casein,
and Westmead
DMBA8 nonmetastatic CDNA1 (WDNM) levels were not
much affected (Fig.
4Cd and data not shown). Similar decreases
in WAP, but not in other
milk proteins, were observed in the mammary
glands of STAT5a-deficient
mice (
17). WAP expression was partially
recovered on days 2 and 3 (Fig. 4Ce, lanes 5 and 6). A similar
recovery in the WAP level
was observed in STAT5a single-knockout
mice, suggesting that STAT5
activity remained in CIS1 transgenic
mice (
17,
35). As shown
in Fig. 4Cf and g, neither c-Myc nor
c-Fos expression was affected by
CIS1 overexpression. These two
genes are probably regulated
independently of STAT5 (
9). Thus,
these data suggest that
CIS1 overexpression selectively modulates
STAT5-regulated gene
expression in mammary
glands.
Inhibition of GH- and PRL-dependent STAT5 activation by CIS1 in 293 cells.
To demonstrate a direct inhibition of GH- and PRL-induced
STAT5 activity by CIS1, we used a transient reporter gene assay in 293 cells (18, 40). A STAT5 reporter gene construct was transfected into 293 cells with the GH receptor (Fig. 5A and
B) or the PRL receptor (Fig. 5C) and CIS1
cDNAs. Increased CIS1 expression inhibited STAT5 activation in response
to GH (Fig. 5A) and PRL (data not shown). GH induced STAT5
transcriptional activity in a dose-dependent manner, and CIS1
expression suppressed GH-mediated STAT5 activation (Fig. 5B). At a low
dose of GH (10 ng/ml), the suppression of STAT5 activity by CIS1 was
almost complete, but it was partial at a higher dose of GH (Fig. 5B).
Therefore, the suppression of STAT5 activity was dependent on both
hormone and CIS1 expression levels. CIS1 also strongly suppressed
PRL-induced STAT5 activity (Fig. 5C). When exogenous STAT5a or STAT5b
was coexpressed, CIS1 still could suppress reporter gene activation, but the inhibitory effect of CIS1 became partial (Fig. 5C and data not
shown). Similarly, inhibition of GH-induced STAT5 activation by CIS1
was overcome by STAT5 expression (data not shown). Therefore, the
negative effect of CIS1 was also dependent on STAT5 expression levels.
These data are consistent with our hypothesis that CIS1 suppresses
STAT5 activation by blocking the binding of STAT5 to the receptor
binding sites.
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FIG. 5.
Inhibition of GH- or PRL-dependent STAT5 activation
demonstrated by a reporter gene assay. (A) 293 cells were transfected
with plasmids of the reporter gene (50 ng of plasmid), the
-galactosidase gene (50 ng), the GH receptor cDNA (200 ng), and
increasing amounts of CIS1 expression plasmid (columns 1 and 2, 0 ng;
column 3, 0.01 ng; column 4, 0.05 ng; column 5, 0.1 ng; column 6, 0.5 ng; column 7, 1 ng; column 8, 5 ng). After transfection, cells were
incubated in the presence (+) or absence () of 50 ng of GH/ml for
6 h, and luciferase activity was then measured. Data normalized
with the -galactosidase activity from triplicate experiments are
shown. (B) 293 cells were transfected with the reporter gene, the
-galactosidase gene, and the GH receptor cDNA in the presence (+) or
absence () of CIS1 cDNA (10 ng). After transfection, cells were
incubated with the indicated concentrations of GH for 6 h. (C) 293 cells were transfected with the reporter gene, the -galactosidase
gene, the PRL receptor (200 ng), and CIS1 cDNA (100 ng) in the absence
() or presence of STAT5b cDNA (+, 5 ng; ++; 10 ng). After
transfection, cells were incubated with 50 ng of PRL/ml for 6 h,
and luciferase activity was then measured.
|
|
T-cell development in CIS1 transgenic mice.
Next, we evaluated
the role of CIS1 on the development of T cells, since recent STAT5 gene
knockout studies revealed an important role of STAT5 in these cells.
Tg2 and Tg3 mice expressed Myc-CIS1 in the spleen, while Tg1 expressed
it at lower levels (Fig. 6A). The
expression levels of endogenous and exogenous CIS1 mRNA (Fig. 6B) were
compared in unstimulated splenic T cells (lanes 1 and 3) and splenic T
cells stimulated (lanes 2 and 4) with anti-TCR and anti-CD28 MAbs
(TCR-plus-CD28 stimulation). Stimulation was confirmed by the induction
of IL-2 receptor chain mRNA (Fig. 6Bb). In unstimulated T cells of
Tg3 mice, myc-CIS1 mRNA was expressed at high levels, (Fig.
6Ba, lane 3), while endogenous CIS1 was undetectable in wild-type mice
(lane 1). However, the endogenous CIS1 mRNA was strongly induced by
TCR-plus-CD28 stimulation in both wild-type and Tg3 mice (Fig. 6Ba,
lanes 2 and 4). The level of endogenous CIS1 was comparable to that of
exogenous Myc-CIS1 in Tg3 mice (Fig. 6B, lane 4).
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FIG. 6.
Expression of Myc-CIS1 protein in the spleen (A), CIS1
mRNA levels in splenic T cells (B), and development of NK cells (C) and
T cells (D). (A) The spleen extract (10 µg/lane) from each
transgenic line was immunoblotted with an anti-Myc antibody (a) and an
anti-STAT5 antibody (b). (B) Total RNA was extracted from unstimulated
() splenic T cells (2.5 × 106 cells/ml) (lanes 1 and 3) from wild-type (WT) and CIS1 transgenic (Tg3) mice or from T
cells after stimulation with anti-TCR and anti-CD28 for 36 h (+)
(lanes 2 and 4). Endogenous CIS1, exogenous Myc-CIS1, and
IL-2 receptor chain (IL-2R) mRNA levels were analyzed by
Northern hybridization (a and b). 28S and 18S RNA levels are shown in
panel c. (C and D) Liver and spleen cells from WT, Tg2, and Tg3 mice
were stained with an anti-CD3 MAb and anti-NK1.1 (C) or anti- TCR
(D), then analyzed by flow cytometry. Numbers represent the percentages
of cells in the gated populations. The experiments were performed
several times and with different litters, and essentially the same
results were obtained.
|
|
Transgenic expression of CIS1 did not significantly affect the number
of thymocytes and splenocytes (data not shown). The
CIS1 transgenic
mice showed slight reductions in the numbers of
CD8
+SP
cells in the spleen, while the numbers of CD4
+SP cells were
unaffected (data not shown). The most drastic difference
in splenic T
cells between wild-type and transgenic mice was the
number of
T
cells (Fig.
6D). In transgenic mice, the numbers
of NK1.1
+
CD3
(NK) and NK1.1
+ CD3
+ (NKT)
cells were decreased in the spleen, bone marrow, and liver
(Fig.
6C and
data not shown). Especially, the number of liver
NKT cells in Tg3 mice
was about 10% of that in wild-type mice
(Fig.
6C). We did not see much
reduction in the numbers of
T cells and NK T cells in Tg1 mice,
probably reflecting low exogenous
CIS1 expression levels (data not
shown). These data indicate that
overexpression of CIS1 suppresses
T-cell, NK cell, and NK T-cell
development.
Decreased response of T cells to IL-2 in transgenic mice.
We
evaluated the effect of CIS1 overexpression on T-cell responsiveness to
IL-2 stimulation, since IL-2 strongly induces CIS1 expression
(41). Using STAT5a or -b single-knockout mice, Nakajima et
al. (23) and Imada et al. (11) reported that the
IL-2 receptor chain has been shown to be partly regulated by STAT5.
Therefore, we measured the increase in the IL-2 receptor chain
level in response to IL-2 by following their methods. First splenocytes from wild-type and CIS1 transgenic mice were preactivated with PMA and
concanavalin A (ConA) for 24 h, and then cells were cultured in
the presence of IL-2 for an additional 40 h. At each stage, the
IL-2 receptor chain expression was examined with a
fluorescence-activated cell sorter (Fig. 7A). PMA-plus-ConA treatment
induced the IL-2 receptor chain similarly in wild-type and
transgenic mice (Fig. 7Ab and e). Further
stimulation with IL-2 resulted in the appearance of a population with a
much higher level of IL-2 receptor chain expression in wild-type
mice (Fig. 7Ac). However, the appearance of such an IL-2 receptor chain-high population was retarded in CIS1 transgenic mice (Fig. 7Af).
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|
FIG. 7.
IL-2 responses of splenocytes from wild-type (WT) and
CIS1 transgenic (Tg3) mice. (A) Flow-cytometric analysis of
IL-2-receptor chain (IL-2R) expression. Splenocytes (5 × 106 cells/ml) were stimulated with 10 µg of ConA/ml and
50 ng of PMA/ml for 24 h, then washed twice with PBS, and finally
cultured with 100 Unit of IL-2/ml for an additional 40 h. Cells
were stained with an FITC-conjugated anti-IL-2R MAb before
stimulation (a and d), after stimulation with ConA and PMA (b and e),
and after further stimulation with IL-2 (c and f). (B) Naive splenic T
cells from WT or Tg3 mice were preactivated with a plate-bound anti-TCR
MAb. After 36 h, the T cells were cultured with various
concentrations of IL-2 in the presence or absence of PMA (50 ng/ml) for
48 h. After pulse-labeling with 1 µCi of
[3H]thymidine for an additional 8 h, the
radioactivity incorporated into cells was measured.
|
|
We examined the effect of CIS1 expression on the IL-2-induced
proliferation of T cells. CD4
+ T cells were enriched from
the spleen and preactivated for 36
h with a plate-bound anti-TCR
MAb. After harvest and washing,
T cells were incubated with various
amounts of IL-2 in the presence
or absence of PMA. As shown in Fig.
7B,
T cells from CIS1 transgenic
mice constantly presented a partial
reduction in IL-2-dependent
proliferative responses. The responses of T
cells from CIS1 transgenic
mice were about 50% of those from their
wild-type littermates
at low doses of IL-2 in the presence of PMA.
However, the responses
to IL-2 were similar in wild-type and transgenic
mice at high
concentrations of IL-2.
Next, we examined the effect of transgenic CIS1 on the induction of
STAT5-responsive genes (the endogenous CIS1 and oncostatin
M [OSM]
[
41,
42] genes) as well as the phosphorylation of
STAT5 in splenic T cells. As shown in Fig.
8Aa and b, IL-2 strongly
induced the
expression of endogenous CIS1 and OSM in wild-type
mice (lane 2).
However, induction of these genes was partially
suppressed in CIS1
transgenic mice (Fig.
8A, lane 4), while c-
myc was equally
induced by IL-2 in wild-type and transgenic mice (Fig.
8Ac), suggesting
that CIS1 selectively inhibited the induction
of STAT5-responsive
genes.
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FIG. 8.
Induction of STAT5-responsive genes and tyrosine
phosphorylation of STAT5 in response to IL-2 in T cells from wild-type
(WT) and CIS1 transgenic (Tg3) mice. (A) Splenic T cells were
preactivated with a plate-bound anti-TCR MAb for 36 h and cultured
without stimulation for 6 h. Then cells were stimulated without
() or with (+) 100 U of IL-2/ml for 60 min. Endogenous CIS1,
exogenous Myc-CIS1, OSM, c-Myc, and glyceraldehyde-3-phosphate
dehydrogenase (G3PDH) mRNA levels were analyzed by Northern
hybridization. (B) Splenic T cells preactivated with an anti-TCR MAb
were stimulated with the indicated concentrations of IL-2 for 10 min;
then the total-cell extracts were resolved by SDS-PAGE and
immunoblotted with an anti-STAT5 (a) or an anti-pY-STAT5 (b) antibody.
(C) Splenocytes were stimulated without () or with (+) 100 ng of
LIF/ml for 30 min, then immunoblotted with an anti-STAT3 (a) or an
anti-pY-STAT3 (b) antibody.
|
|
IL-2-dependent STAT5 phosphorylation was markedly reduced in T cells
preactivated with anti-TCR from CIS1 transgenic mice
compared with
those from their wild-type littermates (Fig. 8Bb).
On the other hand,
LIF-induced tyrosine phosphorylation of STAT3
was not affected by CIS1
overexpression (Fig. 8Cb). These data
indicate that the overexpression
of CIS1 specifically inhibits
STAT5 activation, thereby suppressing
IL-2-induced IL-2 receptor
chain expression, proliferation, and
STAT5-responsive gene
expression.
CIS1 overexpression promotes differentiation into Th2 cells.
We observed that stimulation of T cells with the antibodies against TCR
and CD28 markedly enhanced the expression of CIS1 mRNA (Fig. 6B).
Therefore, we addressed the question of whether constitutive expression
of CIS1 affects the development of helper T cells induced by
TCR-plus-CD28 stimulation in the presence or absence of IL-4. After 5 days, the primed CD4+ T cells were restimulated with the
anti-TCR MAb for 6 h, and the Th1 and Th2 differentiation profiles
were evaluated by intracellular IL-4 and IFN- staining
(2). In B6C3-F1 wild-type mice, this stimulation generated
quite a low IL-4-positive (Th2) population, while IFN--positive
(Th1) cells were predominant (Fig. 9A).
The presence of IL-4 in the initial activation stage was required for
the development of Th2 cells in wild-type mice (Fig. 9A). The
transgenic expression of CIS1 apparently influenced the proportions of
Th1 and Th2 cells. The number of Th1 cells consistently decreased in
CIS1 transgenic mice to almost two-thirds of that in their wild-type
littermates (Fig. 9B). In contrast, Th2 development was significantly
increased by CIS1 overexpression, and the proportion of Th2 cells in
transgenic mice was twice as high as that in wild-type mice (Fig. 9B).
The increase in Th2 development in transgenic mice was confirmed by an
enzyme-linked immunosorbent assay. Precultured T cells from CIS1
transgenic mice showed the ability to secrete three-times-higher
amounts of IL-4 than wild-type mice (data not shown). Taken together,
CIS1 profoundly skews helper T-cell development and shifts the balance
from Th1 dominant to Th2 dominant.
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FIG. 9.
Th1/Th2 differentiation of CD4+ naive T
cells from wild-type (WT) and CIS1 transgenic mice. (A) Naive T cells
were stimulated with anti-TCR (30 µg/ml) plus anti-CD28 (1 µg/ml)
MAbs in the presence (lower panels) or absence (upper panels) of 10 ng
of IL-4/ml. After 5 days, the primed CD4+ T cells were
restimulated with an anti-TCR MAb for 6 h in the presence of 4 µM monensin, and then cells were analyzed by flow cytometry followed
by staining with FITC-conjugated anti-IFN- and PE-conjugated
anti-IL-4. Numbers represent the percentages of cells in the gated
populations. (B) Percentages of the Th1 and Th2 populations from five
independent experiments. Bars, standard errors.
|
|
| |
DISCUSSION |
Comparison of the phenotypes of CIS1 transgenic and STAT5 knockout
mice.
We have shown that CIS1 is induced by several cytokines that
activate STAT5 and that it binds to the EPO and IL-3 receptors, thereby
partially suppressing STAT5 activity. We have also shown that CIS1
overexpression suppressed EPO-, GH-, and PRL-induced STAT5 activation
by using a reporter gene assay (18, 19) (Fig. 5). The
inhibitory effect of CIS1 depends on the cytokine concentration and the
CIS1 level as well as the STAT5 level (Fig. 5). In this study, using
transgenic mice, we demonstrated that CIS1 overexpression actually
inhibited several cytokine- or hormone-mediated STAT5 functions in
vivo. We confirmed phenotypes by using three or two independent
transgenic lines, and the extent of phenotypes was dependent on the
expression levels of transgenic CIS1.
The phenotypes observed in CIS1 transgenic mice are strikingly similar
to those of STAT5a and/or STAT5b knockout mice. CIS1
transgenic mice
exhibited defects in growth, mammary-gland development,
and T-cell
response and NK cell development. This is a mixture
of the phenotypes
found in STAT5a and STAT5b single-knockout mice
or is close to that of
STAT5a,b double-knockout mice. For example,
STAT5a knockout results in
retardation of mammary-gland development
but not of growth, while in
STAT5b knockout mice, the body growth
of males was retarded but
mammary-gland development was not much
affected (
35). NK
cell development was more severely affected
in STAT5b null mutant mice
than in STAT5a null mutant mice (
11).
It has been reported
that the major STAT5 form activated by GH
in the liver is STAT5b
(
25,
35), while the major form activated
in mammary glands
is STAT5a (
3); such a different localization
may account for
the separate effects of STAT5a and -b single-gene
knockouts in mice, as
suggested by Teglund et al. (
35). Since
CIS1 can suppress
both STAT5a and STAT5b (data not shown), the
phenotype of CIS1
transgenic mice could be similar to that of
STAT5a,b double-knockout
mice.
However, there are some differences between CIS1 transgenic mice and
STAT5a,b double-knockout mice. For example, CIS1 transgenic
female mice
are fertile, while STAT5a,b double-mutant female mice
are infertile and
have altered ovarian development (
35). T cells
from STAT5a,b
double-knockout mice fail to proliferate in response
to anti-TCR and
IL-2 (
21), while suppression in CIS1 transgenic
mice is only
partial. There is a detectable reduction in the number
of hematopoietic
colonies induced in response to IL-3, IL-5, and
GM-CSF in STAT5a,b
double-knockout mice (
35), while we did not
see any
difference in hematopoietic colony formation in vitro
in CIS1
transgenic mice (data not shown). These differences are
probably due to
the partial inhibitory effect of CIS1 on STAT5a
and -b, which depends
on cytokine concentrations as well as CIS1
and STAT5 expression levels.
Nevertheless, the similarity of the
phenotypes of CIS1 transgenic mice
to those of STAT5 knockout
mice strongly supports our notion that CIS1
is a specific inhibitor
of
STAT5.
Comparison with other CIS family member transgenic mice and
functional overlapping.
We have reported on other CIS1-related
proteins, including JAB and CIS2 to CIS6 (6, 18, 28, 32). We
have tried to create JAB, CIS2, CIS3, and CIS4 transgenic mice under
the control of the -actin promoter, which is also used for CIS1
transgenic mice. We have established CIS2 and CIS4 transgenic mice with
levels of protein expression comparable to that of CIS1. However, we have not seen any defects so far in growth, lactation, and T-cell development in these transgenic mice. Therefore, the phenotype of CIS1
transgenic mice is not due to overexpression of an SH2 domain; rather,
CIS1 seems to specifically inhibit STAT5 activity. Consistent with this
notion, CIS2 and CIS4 did not inhibit EPO-dependent STAT5 activation
(18). The target molecules of CIS2 and CIS4 have not been
identified as yet; however, detailed analysis of the phenotype of
transgenic mice will give us a clue to the functions of CIS2 and CIS4.
On the other hand, we could not obtain JAB and CIS3 transgenic mice
using the -actin promoter. These two molecules can bind to JAKs
directly and strongly inhibit kinase activity. Therefore, JAB and CIS3
overexpression is probably embryo lethal, like JAK2 gene disruption.
The inhibitory effect of CIS1 on cytokine-dependent STAT5 activation is
partial after stimulation with very high doses of
cytokines (see Fig.
5). On the other hand, CIS3 can strongly inhibit
PRL-dependent STAT5
activation, and the inhibitory effect of CIS3
on STAT5 activation was
much more profound than that of CIS1 (
8).
In this sense, it
is notable that CIS3 is expressed at high levels
in mammary glands.
Furthermore, CIS3 expression was partially
suppressed in CIS1
transgenic mice, suggesting that these two
molecules are also regulated
by STAT5 in this tissue. We recently
found that CIS3 binds to the same
cytokine receptors as CIS1,
including the EPO receptor and the IL-2
receptor
chain. Moreover,
the binding region of the EPO receptor to
CIS3 is similar to that
to CIS1. Therefore, CIS3 may have an
overlapping function with
CIS1.
Roles of CIS1 and STAT5 in T- and NK cell development.
STAT5a
and STAT5b have been shown to play an important role in T-cell and NK
cell development (11, 21, 23). T cells from STAT5b
single-knockout mice do not respond to anti-TCR stimulation, and
expression of the IL-2 receptor chain is diminished
(11). NK cell development is also impaired in
STAT5b-deficient mice. The response of T cells to IL-2 was also
partially suppressed in CIS1 transgenic mice. However, these defects in
CIS1 transgenic mice and STAT5a knockout mice are modest compared to
those in STAT5b single-knockout or STAT5a,b double-knockout mice. Thus, CIS1 transgenic mice rather resemble STAT5a knockout mice in the response of T cells to IL-2. Interestingly, the number of T cells was decreased in CIS1 transgenic mice as in STAT5a-deficient mice, while these cells were normal in STAT5b-deficient mice. In
contrast, the decreases in the numbers of NK cells and NKT cells in
CIS1 transgenic mice were more drastic than those found in STAT5b
knockout mice, while the number of NK cells was not much affected in
STAT5a knockout mice (11, 23). Therefore, again, defects of
lymphocytes in CIS1 transgenic mice are a mixture of those found in
mice lacking either STAT5a or STAT5b. Such different effects of STAT5a
and STAT5b may be explained by their distinctive expression patterns in
specific lymphocyte populations and by different target genes of the
two proteins. Importantly, STAT5a,b double-knockout mice develop
splenomegaly and have T cells with an activated phenotype, which were
not seen in mice lacking STAT5a or STAT5b alone or in our CIS1
transgenic mice. This is probably because T cells in CIS1 transgenic
mice as well as STAT5a or -b signal knockout mice retain some STAT5 activity.
It is notable that endogenous CIS1 was normally induced by
TCR-plus-CD28 stimulation in CIS1 transgenic mice (Fig.
6B, lane
4),
while it was marginally induced through TCR-plus-IL-2 stimulation
(Fig.
8A, lane 4). The IL-2 receptor
chain was also equally
induced by
TCR-plus-CD28 stimulation in wild-type and Tg3 mice
(Fig. 6Bb). This
may be because signals from TCR and CD28 induced
the endogenous CIS1
gene and the IL-2 receptor
chain without
using STAT5 or because
CIS1 did not inhibit TCR- and CD28-dependent
signals, including
TCR-mediated STAT5 activation (
38).
In this study, we found that Th1/Th2 development was altered by CIS1
constitutive expression. Helper T-cell development has
not been
examined in STAT5a or -b single-knockout mice. Th1/Th2
development has
been shown to be profoundly regulated by IL-4-STAT6
and IL-12-STAT4
systems. However, it has been well known that
CD28 costimulation
induces the secretion of large amounts of IL-2
in naive T cells, and
IL-2 signals have been shown to be required
for the proliferation and
maintenance of Th1 cells. Therefore,
Th2 cells may develop in CIS1
transgenic mice, since IL-2 signals
would be blocked by CIS1
overexpression. On the other hand, Moriggl
et al. (
21)
showed that STAT5a,b double-deficient lymphocytes
consistently produced
less IL-2 and more IFN-
. As suggested by
the authors, high IFN-
production in the T cells of double-knockout
mice may be due to
predominance of a CD62L
CD44
+ population.
However, as an alternative explanation, an increase
in the Th1-type
cytokine level in STAT5a,b double-knockout mice
may be attributed to
the lack of CIS1 expression, since CIS1 was
not expressed in T cells
from STAT5a,b double-knockout mice. This
is consistent with our data of
Th2 increase in CIS1 transgenic
mice. Our data raise the interesting
possibility that CIS1 rather
than STAT5 plays an important role in
regulating Th1/Th2
balance.
Mechanism of STAT5 inhibition by CIS1.
CIS1 protein has been
shown to associate with the tyrosine-phosphorylated EPO and IL-3
receptors (41). We recently found that CIS1 protein can also
bind to the PRL receptor and the IL-2-receptor chain but not to
gp130, a signal-transducing subunit of the IL-6 receptor. Selective
binding of CIS1 to the receptors which activate STAT5 may explain the
selective negative effect of CIS1 on STAT5 activation.
However, the molecular mechanism of STAT5 inhibition by CIS1 has not
been clarified yet. We reported previously that CIS1
binds to the
region of the EPO receptor containing the second
tyrosine residue
(Y401) (
37). One of the mechanisms is simply
masking the
STAT5 binding sites on the receptor. This model is
supported by our
observation that overexpression of STAT5 can
overcome the negative
effect of CIS1 (Fig.
5C). The other possibility
is that CIS1
accelerates the degradation of the receptor-CIS1
complex by the
ubiquitin-proteasome pathway (
37). Since proteasome
inhibitors stabilize the tyrosine-phosphorylated form of the EPO
receptor as well as STAT5, and since CIS1 is rapidly ubiquitinated,
this model is an attractive hypothesis. However, this model cannot
explain the selective negative effects of CIS1 overexpression
on STAT5,
since CIS1 overexpression did not affect EPO or IL-2-dependent
c-
myc induction (
19) (Fig.
8). Further study is
necessary to
elucidate the mechanism of STAT5 inhibition by
CIS1.
| |
ACKNOWLEDGMENTS |
We thank H. Ohgusu and M. Sasaki for excellent technical
assistance, M. Chikushi for preparing the manuscript, A. Gertler for
donating purified GH and PRL, W. Doppler for providing PRL receptor
cDNA, and T. Kitamura for making available to us STAT5a and -b cDNAs.
Part of this work was supported by grants from the Ministry of Science,
Education, and Culture of Japan, the Uehara Memorial Foundation, the
TORAY Research Foundation, the Naito Memorial Foundation, the Sumitomo
Foundation, and the Kanae Research Foundation.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
Life Science, Kurume University, Aikawa-machi 2432-3, Kurume 839-0861, Japan. Phone: 81-942-37-6313. Fax: 81-942-31-5212. E-mail:
yosimura{at}lsi.kurume-u.ac.jp.
| |
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Molecular and Cellular Biology, September 1999, p. 6396-6407, Vol. 19, No. 9
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Abstract
Introduction
