Cytokine Receptor Common 
Subunit-mediated STAT5 Activation
Confers NF-
B Activation in Murine proB Cell Line Ba/F3 Cells*
Tetsuya
Nakamura
,
Rika
Ouchida,
Tsunenori
Kodama,
Toshiyuki
Kawashima§,
Yuichi
Makino,
Noritada
Yoshikawa,
Sumiko
Watanabe¶,
Chikao
Morimoto,
Toshio
Kitamura§, and
Hirotoshi
Tanaka
From the Division of Clinical Immunology,
§ Division of Hematopoietic Factors, Advanced Clinical
Research Center, and ¶ Department of Molecular and
Developmental Biology, Institute of Medical Science, University of
Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
Received for publication, October 12, 2001, and in revised form, December 5, 2001
 |
ABSTRACT |
The cytokine receptor common 
subunit
(c) transmits intracellular signals upon binding
ligand such as granulocyte-macrophage colony-stimulating factor or
interleukin-3 (IL-3); however, transcriptional regulation under the
control of signaling events downstream of the c is not
fully understood. Using murine Ba/F3 cells, here we demonstrate that
the c-mediated signals stimulate NF-
B-driven gene
expression of not only the reporter construct but also endogenous target genes such as IL-6. Analyzing the effects of several
inhibitors or mutant receptors revealed that this NF-B activation is
mediated neither by MEK/ERK/MAPK nor by the phosphatidylinositol
3-kinase pathway but by STAT5. Overexpression experiments of the
wild-type or constitutive active form of STAT5 further confirmed this
notion. In addition, STAT5-dependent NF-B activation is
mediated not through an inducible nuclear translocation but via
up-regulation of both DNA binding activity and transactivation
potential of NF-B. Furthermore, we also show that as yet undefined
humoral factor(s) may be involved in this NF-B activation process.
Taken together, we may propose that cytokine receptor-mediated STAT5 activation and expression of its target genes culminates in a unique
mode of NF-B activation and gene expression.
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INTRODUCTION |
Granulocyte-macrophage colony-stimulating factor
(GM-CSF)1 and interleukin-3
(IL-3) play important roles in regulating multiple cellular functions
such as proliferation, differentiation, and survival in various
hematopoietic cell lineages and their precursors (1). These cytokines
bind to their cognate receptors and trigger a cascade of signaling
events leading to various biological responses. The receptors for
GM-CSF (GMR) and IL-3 (IL-3R) are composed of two subunits, the
cytokine-specific 
and the common subunit (c),
the latter of which is also shared by the receptor for IL-5 (2). Both
subunits belong to the type I cytokine receptor superfamily, and the
c, having a relatively large cytoplasmic domain, plays a
pivotal role in downstream signal transduction (2). These receptors
lack intrinsic kinase activity but interact with and activate Janus
kinase 2 (JAK2) in response to binding ligand (3). Subsequently, the
cytoplasmic domain of the c becomes
tyrosine-phosphorylated and then cue multiple signaling pathways (4, 5)
including the mitogen-activated protein kinase (MAPK) pathway and the
phosphatidylinositol 3-kinase (PI3K) pathway (6, 7). In
addition, at the transcriptional level, not only expression of some
immediate early genes such as c-fos, c-jun,
c-myc, egr-1, cis, and
pim-1 (6, 8, 9) but also the functional regulation of
transcription factors such as STAT5, AP-1, and CREB (9-12) is known to
be achieved by these cytokine receptors.
The STAT proteins are activated upon various cytokine stimulations and
play a central role in following the transcriptional regulation of gene
expression (13, 14). Among seven known members of STAT family, both
STAT5A and STAT5B, closely related isoforms, are the most predominant
members activated by the c (10, 11). Upon cytokine
stimulation, JAK2 phosphorylates STAT5 on a tyrosine residue near the
C-terminal transactivation domain in the cytoplasm (15). The
phosphorylated STAT5 proteins dimerize and translocate into the
nucleus, where they bind to specific DNA elements and thereby activate
target gene expression (10, 11). Using murine
IL-3-dependent Ba/F3 cells, we have reported previously
that STAT5 participates in growth-promoting signals downstream of the
c (16-18). Moreover, we have shown that STAT5 is a key
regulator of not only proliferation but also differentiation or
apoptosis in these cells (19). In particular, pim-1,
JAB/SOCS-1/SSI-1, and p21WAF1/Cip1, all of which are
transcriptionally regulated by STAT5, are suggested to be
involved in a variety of biological outcomes induced by STAT5 (19). Our
knowledge of the gene expression profile regulated by STAT5, however,
has been extremely limited. A recent study using microarray analysis
revealed that more than 300 genes are regulated at the transcriptional
level by IL-3 signaling in murine pro-B cells (20), raising the
possibility that more complicated mechanisms than described previously
are operated in these cytokine- or STAT5-dependent
biological responses.
It was recently reported that several growth factors, including GM-CSF
(21) and IL-3 (22), could induce the activation of another
transcription factor NF-B in cells of hematopoietic lineages,
although underlying mechanisms have remained unclear. NF-B was
originally described as a regulator of immune and inflammatory responses. NF-B consists of a dimer from five related proteins, most
typically a heterodimer composed of p65/RelA and p50 subunits (23-25).
The regulation of NF-B is achieved through interaction with a family
of inhibitory protein known as IB that binds to NF-B and
sequesters it in the cytoplasm (26). Once cells are stimulated with
inducers such as tumor necrosis factor- (TNF) and IL-1, two
serine residues of the IB protein are phosphorylated by IB
kinases (27, 28). Phosphorylation of IB targets it for
ubiquitination and subsequent degradation by the 26S proteasome and
renders the nuclear localization signal of NF-B unmasked (29,
30). Then NF-B translocates from the cytoplasm into the nucleus and
regulates the transcription of target genes. In addition to this
"classical" milieu, recent reports have suggested that alternative
pathways lead to NF-B activation through several nuclear mechanisms
(31-35). As well as its well established role in activating the
transcription of genes involved in immunological responses, it is
indicated that NF-B is one of the central mediators of hematopoiesis
(36). Therefore, it appears to be of importance to elucidate the
linkage between the GM-CSF or IL-3 signaling and NF-B activation for
a better understanding of biological responses under the control of
these cytokines.
In the present study, we demonstrated that in Ba/F3 cells, GM-CSF as
well as IL-3 activates NF-B-dependent transcription, and
STAT5 is essential for this process. Of note, STAT5 affected neither
IB degradation nor nuclear translocation of NF-B. In contrast, we
indicated that STAT5 increases not only the DNA binding activity but
also the transactivational potential of NF-B, possibly through a
mechanism involving STAT5-dependent synthesis of as yet
undetermined humoral factor(s).
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EXPERIMENTAL PROCEDURES |
Cell Culture--
A murine (m) IL-3-dependent proB
cell line, Ba/F3 (37), was maintained in RPMI 1640 (Sigma)
medium containing 10% fetal calf serum (Medical & Biological
Laboratories, Nagoya, Japan), 4 ng/ml mIL-3 (Pepro Tech EC, London,
England), 100 units/ml penicillin, and 100 µg/ml streptomycin.
Various Ba/F3 cell clones stably expressing human (h) GMR together
with hGMRc (Ba/F3-wild) or hGMRc mutant Y6 or Fall (Ba/F3-Y6 or Ba/F3-Fall, respectively) have been described elsewhere (16). These engineered cells were grown in medium in the
presence of 500 µg/ml G418 (Sigma). Another clone of Ba/F3 cells in
which STAT5A1*6 is stably transfected (Ba/F3-STAT5A1*6), as well as a
murine myelomonocytic cell line (WEHI 3B cells), was maintained with
the same medium but without IL-3 (tentatively termed as depletion
medium). In all experiments, cells were extensively washed with the
depletion medium three times and stimulated with either 4 ng/ml mIL-3,
5 ng/ml hGM-CSF (kindly provided from Kirin Brewery, Tokyo, Japan), or
indicated concentrations of mTNF (Pepro Tech EC), mIL-6 (Sigma),
m-oncostatinM (OSM, Sigma), or mIL-1 (Pepro Tech EC) or left
nonstimulated. Conditioned medium was prepared by culturing either
parental Ba/F3 or Ba/F3-STAT5A1*6 cells at a density of 1 × 107 cells/30 ml for 24 h in the depletion medium. The
culture supernatant was subsequently collected, cleared by
centrifugation (1,000 × g for 20 min), and freshly
used as a conditioned medium for stimulating cells.
Plasmids--
The reporter plasmids NF-B-Luc (formerly
pNFBHL) and AP-1-Luc (formerly pAP-1HL), in which expression of the
luciferase gene is under the control of transcription factors
NF-B and AP-1, respectively, were described elsewhere (38). A
-casein promoter-inducible luciferase reporter construct,
-casein-Luc (pZZ1) (39), and an expression plasmid,
pXM-STAT5
750VP16JAK2, encoding a fusion protein described in Ref. 40
(see the legend for Fig. 7), were kindly gifts of Dr B. Groner
(Tumor Biology Center, Freiburg, Germany). To construct the
wild-type (WT) STAT5A expression vector pCMX-WT STAT5A, an
EcoRI-NotI fragment containing cDNA for
mSTAT5A was excised from a retrovirus vector pMX-STAT5A (18), filled with the Klenow fragment, and inserted into EcoRV site of
pCMX (41). A pCMX-STAT5A1*6, an expression vector encoding a
constitutive active mutant form of STAT5A, was constructed according to
the same strategy as above using pMX-STAT5A1*6 (18). A
pCMX-STAT5A749 encoding a mutant of STAT5A in which
C-terminal 44 amino acids are deleted was derived by exchanging the
NsiI and NheI fragments of pCMX-WT STAT5A with
the PCR-generated fragment: primers used in the PCR reaction
were 5'-GAGTTCGTCAATGCATCCAC-3' (sense primer) and
5'-AGTCAGTCGCTAGCTGAGCCATCTTGGTCAAGGAC-3'
(antisense primer). Underlined sequences indicate recognition sites
for NsiI for the sense primer or NheI for the
antisense primer. Bold sequences indicate the introduced stop codon. A
pCMX-STAT5AVVV encoding a DNA-binding defective mutant of STAT5A (42)
(see the legend for Fig. 7) was generated by the use of sequential PCR
reactions. Primers used here were
5'-AGAAGCAGCCTCCTCAGGTC-3' (sense primer-1), 5'- ATGGACGATAGCGGCCGCAGGGAGGGACAG-3'
(antisense primer-1),
5'-TCCCTCCCTGCGGCCGCTATCGTCCATGGC-3' (sense primer-2), 5'-GTTGTAGTCCTCGAGGTGGT-3' (antisense
primer-2), and two independent PCR reactions were carried out with
either set of primers, respectively, using a pCMX-WT STAT5A as a
template. The second step of the PCR reaction was performed with the
outer primers, sense primer-1 and antisense primer-2, using combined products of the first round PCR as templates. The Bsu36I and
XhoI fragment within the STAT5A coding region of a pCMX-WT
STAT5A was replaced with the PCR-amplified fragments. Underlined
sequences are sites for Bsu36I (sense primer-1) and
XhoI (antisense primer-2), respectively, and the bold
letters indicate introduced mutations at codons 466, 467, and 468. A
Gal4-responsive luciferase reporter tk-Gal4px3-Luc was described
elsewhere (43). To construct the chimeric plasmid for NF-B p65 and
the DNA binding domain of Gal4 (amino acids 1-147),
PCR-generated fragments containing cDNA encoding either the N-terminal
half (amino acids 1-285) or C-terminal half (amino acids 286-549) of
m-p65 were cloned into EcoRI and EcoRV sites of
pCMX-Gal4 (44) in-frame. Primers used here were
5'-AGTCAGTCGAATTCATGGACGAACTGTTCCCC-3'(sense primer)
and
5'-AGTCAGTCGATATCTGATTCCATGGGCTCACTGAA-3'(antisense primer) for pCMX-Gal4-p651-285 and
5'-GTGAGCCCATGGAATTCCAG-3'(sense primer) and
5'-AGTCAGTCGATATCTGAGGAGCTGATCTGACTCAG-3' (antisense primer) for pCMX-Gal4-p65286-549,
respectively, and the PCR reaction was performed using pCAGGS-m-p65 (a
gift from Dr. H. Handa, Tokyo Institute of Technology, Tokyo, Japan) as
a template. Underlined sequences indicate recognition sites of
EcoRI for sense primers or EcoRV for antisense
primers. Bold sequences indicate the introduced stop codon. All
plasmids constructed as above were verified by sequencing.
Transient Transfection and Reporter Assays--
Plasmids were
transiently transfected into Ba/F3 cells or WEHI 3B cells by
electroporation as described previously (16). After transfection, cells
were cultured in appropriate conditions, and cellular extracts were
isolated as described below. When indicated, either PD98059 (Biomol
Research Laboratories, Plymouth Meeting, PA) or wortmannin (Sigma) was
added in the culture medium 1 h prior to the addition of
cytokines. Anti-mouse TNF antibodies (R&D systems, Minneapolis, MN)
were also added in the culture medium 2 h prior to the cellular
stimulation. For reporter gene assay, cells were harvested and lysed
by three cycles of freezing and thawing after 12 h (Ba/F3
cells) or 18 h (WEHI 3B cells) of cellular stimulation. Cell
lysates were subjected to luciferase assays, and relative light units
(RLU) were normalized to the protein amount determined with protein
assay reagent (Pierce) according to the manufacturer's instructions.
Total amounts of plasmid DNA were adjusted to 10 µg/transfection
using pGEM 7Z (Promega, Madison, WI).
RNA Extraction and Northern Blotting--
Total RNA was
extracted by the acid guanidine method using TRIzol Reagent
(Invitrogen Corp., Carlsbad, CA). Twenty µg of total RNA was
subjected to 1% agarose/formaldehyde gel electrophoresis and
subsequently transferred onto a MAGNACHARGE nylon membrane filter
(Osmonics, Westborough, MA). Equal amounts of loading and the transfer
efficiency of RNA samples were verified by ethidium bromide staining of
28 and 18 S rRNAs on the gel and the membrane. The cDNA probe
corresponding to nucleotides 16-761 of the m IL-6 cDNA was
generated by reverse transcription-PCR from an RNA sample of parental
Ba/F3 cells cultured under usual conditions. The probe was labeled with
[
32P]dCTP by random priming using RediPrime
II (Amersham Biosciences, Inc.) according to the manufacturer's
instructions. Hybridization was carried out under ULTRAHyb conditions
(Ambion Inc., Austin, TX) at 42 °C overnight.
Preparation of Cell Extracts--
For preparation of whole cell
extracts, 1 × 106 cells were washed with
phosphate-buffered saline twice and incubated on ice for 15 min in
lysis buffer (1% Nonidet P-40, 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.2 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml aprotinin, 1 µM pepstatin, 50 mM NaF, 1 mM
Na3VO4). After centrifugation at 12,000 × g for 20 min in a microcentrifuge, the supernatant was used
as whole cell extracts. Cytosol and nuclear extracts were prepared as
described by Dignam et al. (45) with minor modifications. In
brief, after two cycles of washing in ice-cold phosphate-buffered
saline, 2 × 107 cells were harvested and
centrifuged at 1,000 × g for 5 min in a
microcentrifuge. The cell pellet was resuspended in 80 µl of buffer A
(10 mM Tris-HCl, pH 7.3, 1.5 mM
MgCl2, 10 mM KCl, 1 mM dithiothreitol, 0.4% Nonidet P-40, 1 mM PMSF, 10 µg/ml
aprotinin, 1 µM pepstatin, 50 mM NaF, and 1 mM Na3VO4). After incubation on ice
for 5 min, cell lysates were centrifuged, and the supernatant was used
as cytosol. The nuclear pellet was then resuspended in 75 µl of
buffer C (20 mM Tris-HCl, pH 7.3, 1.5 mM
MgCl2, 484 mM KCl, 1 mM
dithiothreitol, 0.2 mM EDTA, 25% glycerol, 1 mM PMSF, 10 µg/ml aprotinin, 1 µM
pepstatin, 50 mM NaF, and 1 mM
Na3VO4) and incubated for 30 min at 4 °C.
Nuclear debris were pelleted with centrifugation at 12,000 × g for 15 min, and the supernatant was used as nuclear
extracts. Protein concentration was determined using protein assay
reagent (Pierce).
Electrophoretic Mobility Shift Assay (EMSA)--
Ten µg of
protein was subjected to EMSA. The double-stranded oligonucleotide
probes harboring NF-B (top strand, 5'-AATTCAAGGGACTTTCCGCGTC-3'; bottom strand, 3'-GTTCCCTGAAAGGCGCAGCCGG-5') and STAT5 (top strand, 5'-GATCCGAATTCCAGGAATTCA-3'; bottom strand,
3'-GCTTAAGGTCCTTAAGTCTAG-5') binding sequences were annealed and filled
in by BcaBEST DNA polymerase (TaKaRa, Kyoto, Japan) using
[32p]dCTP and unlabeled dNTPs. Proteins were
incubated with 0.7 ng of the radiolabeled probe in 20 µl of the
reaction mixture containing 20 mM HEPES, pH 7.9, 4 mM dithiothreitol, 0.2 mM EDTA, 12% glycerol, and 100 mM KCl in the presence of 2 µg of sheared salmon
sperm DNA (Wako Pure Chemical Industries, Osaka, Japan) for NF-B or 2 µg of poly(dI-dC)(dI-dC) (Amersham Biosciences, Inc.) for STAT5. For competition experiments, a 2-, 5-, or 20-fold excess of unlabeled double-stranded oligonucleotides of NF-B or STAT5 or 20-fold excess
of nonspecific oligonucleotide containing glucocorticoid responsive
element (43) was added prior to the oligonucleotide probe. In
supershift assays, 2 µg of antibodies against either p65 (sc-372),
p50 (sc-1190), p52 (sc-298), RelB (sc-226), c-Rel (sc-70), Bcl-3
(sc-185), or STAT5 (sc-836) (purchased from Santa Cruz Biotechnology,
Santa Cruz, CA) was added to the nuclear extracts for 1 h prior to
the addition of oligonucleotide probe. Samples were then incubated for
15 min at room temperature and electrophoresed on a 4% non-denaturing
polyacrylamide gel in 0.5× TBE (1× TBE is 89 mM Tris
borate, 89 mM boric acid, and 2 mM EDTA)
buffer. Gels were run at 300 V for 2 h at 4 °C, dried, and autoradiographed.
Immunoblotting--
Ten µg of protein was separated in 10%
SDS-polyacrylamide gels and then transferred to polyvinylidene
difluoride membranes. The membranes were blocked in Tris-buffered
saline (TBS)-T (50 mM Tris-HCl, pH 7.6, 200 mM
NaCl, 0.1% Tween 20) with 5% non-fat dried skim milk. The membranes
were probed with either anti-p65 (sc-372), anti-p50 (sc-1190), or
anti-IB (sc-945, Santa Cruz Biotechnology) antibodies at 1:1000
dilution and then incubated with a secondary antibody conjugated to
horseradish peroxidase (Santa Cruz Biotechnology). For analysis of
IB protein, the membranes were first probed with
anti-phospho-IB (Ser-32) antibody (New England Biolabs,
Beverly, MA) at 1:1000 dilution followed by incubation with a secondary
antibody conjugated to horseradish peroxidase. Subsequently, after
detection of proteins, the same membranes were stripped, reprobed for
anti-IB antibody (sc-371, Santa Cruz Biotechnology) at 1:500
dilution, and probed with a secondary antibody. In all experiments,
proteins were visualized with ECL detection systems (Amersham
Biosciences, Inc.) according to the manufacturer's instructions.
Immunocytochemical Analysis--
For immunocytochemical
analysis, Ba/F3-wild cells were collected and washed once with
phosphate-buffered saline, and then 2 × 105 cells
were cytospined to a silane-coated glass slide. Cells were fixed with
3.7% paraformaldehyde in TBS for 20 min at room temperature, permeabilized for 30 min using TBS containing 0.1% Triton X-100 and
3% bovine serum albumin, and washed twice with TBS. Slides were then
treated with 0.1 mg/ml RNase for 1 h at 37 °C. Incubation with
1:1000 dilution of anti-p65 antibody was performed for 1 h at
37 °C followed by washing two times with TBS, and then incubation was carried out for 1 h with anti-rabbit IgG conjugated to
fluorescein (Santa Cruz Biotechnology) at 1:200 dilution. The slides
were rinsed and exposed to propidium iodide for 2 h at room
temperature, and coverslips were mounted for viewing by a laser
scanning confocal microscopy (Fluoview FV500, OLYMPUS, Tokyo, Japan).
| |
RESULTS |
GM-CSF as Well as IL-3 Induces NF-B Activation in Ba/F3 Cells
through Distinct Mechanisms from
MEK/ERK/MAPK or PI3K Pathway--
To
analyze signaling events downstream of the c, we used an
engineered subline of m IL-3-dependent Ba/F3 cells stably
expressing both wild-type and c subunits of
the h GMR (Ba/F3-wild cells, Fig.
1C) (16) because signals
evoked by the hGMR in these cells are able to substitute for those of
endogenous IL-3R. To test whether NF-B-dependent gene
expression is induced by IL-3 or GM-CSF, both Ba/F3-wild and parental
Ba/F3 cells were transiently transfected with an NF-B-inducible
reporter plasmid NF-B-Luc and cultured in the absence or the
presence of these cytokines, and then cellular lysates were assayed for
luciferase activities. As shown in Fig. 1A, treatment with
IL-3 induced nearly 3-fold induction of reporter gene expression in
both of these cells. In contrast, treatment with GM-CSF resulted in
5-fold increase of reporter gene expression only in Ba/F3-wild cells.
These results suggested that not only the endogenous IL-3R but also
transfected hGMR could activate NF-B-dependent gene
expression in Ba/F3 cells and that cells expressing hGMR provide a
suitable tool for analyzing this c-dependent
NF-B activation.
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Fig. 1.
Signals activated by the
c induce NF-B
activation in Ba/F3 cells. Ba/F3-wild cells and parental Ba/F3
cells were transiently transfected with 3 µg of NF-B luciferase
reporter plasmids NF-B-Luc using the electroporation method
(A). Cells were cultured in depletion medium alone or in
medium containing either 5 ng/ml GM-CSF or 4 ng/ml IL-3. After 12 h, cells were lysed and assayed for reporter activity. Luciferase
activities were normalized to RLU per µg of protein. Ba/F3-wild cells
were transiently transfected with NF-B-Luc and cultured in depletion
medium for 1 h with the indicated doses of PD98059 or wortmannin
or left untreated (B). Cells were then stimulated with 5 ng/ml GM-CSF or left unstimulated for another 12 h, and reporter
activities were assayed. Luciferase activities were normalized to RLU
per µg of protein and indicated as fold activation as compared with
those of nontreated cells. The structures of wild-type, Y6, and Fall
mutants of the human GMR are shown (C). and
c are the and common subunits of the GMR,
respectively. Y and F indicate tyrosines and
introduced phenylalanines, respectively, and their amino acid positions
on the c are indicated. The position of each amino acid
residue is also numbered in parentheses as 1 through 8 from the membrane proximal site in order. Ba/F3-wild, -Y6,
and -Fall cells were transiently transfected with NF-B-Luc and
cultured as described in panel A (D). Luciferase
activities were normalized and indicated as fold activation as compared
with those of nontreated Ba/F3-wild cells. All results are the
mean ± S.D. of 3 independent experiments. Parental Ba/F3,
Ba/F3-wild, -Y6, or -Fall cells were cytokine-deprived for 5 h and
then stimulated with 4 ng/ml IL-3 (parental Ba/F3) or 5 ng/ml GM-CSF
(Ba/F3-wild, -Y6, or -Fall) for the indicated time periods, and the
total RNA was extracted (E). Twenty µg of each RNA sample
was subjected to 1% agarose/formaldehyde gel electrophoresis, and then
Northern blot analysis of IL-6 genes was performed
(top) using the [32p]dCTP-labeled probe
corresponding to the nucleotides 16-761 of the mouse IL-6 cDNA.
Equal amounts of loading were verified by ethidium bromide staining of
28 and 18 S rRNAs on the gel (bottom).
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|
As described previously, the c, upon ligand binding,
activates multiple distinct signals such as the MEK/ERK/MAPK cascade, the PI3K pathway, and STAT5 (6, 7, 11). Because it has been revealed
that both the MEK/ERK/MAPK cascade and PI3K pathway activate NF-B in
certain conditions (31, 46-52), the involvement of these pathways in
NF-B activation in our systems was tested using specific inhibitors
for each pathway. As shown in Fig. 1B, neither the
MEK1 inhibitor PD98059 nor PI3K inhibitor wortmannin did affect
GM-CSF-dependent induction of NF-B-responsive reporter gene expression. The bioavailability of PD98059 was verified by its 50 to 60% of inhibitory effect on AP-1-inducible reporter gene activation
in response to GM-CSF (data not shown). Similarly, both PI3K activity
and phosphorylation of Akt, a downstream target of PI3K, were
completely abrogated by wortmannin at the concentrations used in the
present study in Ba/F3 cells (53). These results indicate that NF-B
activation is mediated through neither MEK/ERK/MAPK nor PI3K pathway.
To further characterize the signal(s) responsible for NF-B
activation, we next utilized the sublines of Ba/F3 cells expressing the
mutated c with the intact subunit, Ba/F3-Y6 and
-Fall cells (Fig. 1C). Accumulating data have revealed the essential role of the c in transmitting downstream
signals, most of which are distinctively regulated through the
phosphorylation of eight tyrosine residues on the
c. Ba/F3-Y6 cells, in which only Tyr-750 out of
eight tyrosine residues on the c remains intact, are a
representative clone that activates STAT5 without eliciting MAPK or
PI3K activation in the presence of GM-CSF (16) (Fig. 1C).
Ba/F3-Fall mutant cells, in which all eight tyrosine residues are
mutated to phenylalanines, hardly activate either MAPK, the PI3K
pathway, or STAT5 (16) (Fig. 1C). As shown in Fig.
1D, treatment with IL-3 activated expression of the
NF-B-responsive reporter gene not only in Ba/F3-wild but in Ba/F3-Y6
and -Fall cells as well. In contrast, treatment with GM-CSF stimulated
reporter gene activation in Ba/F3-wild and -Y6 cells but not in
Ba/F3-Fall cells (Fig. 1D). These results suggest that a
certain signal involving neither MAPK nor PI3K pathway but involving
phosphorylation of at least Tyr-750 on the c is
essential for the c-dependent NF-B activation. Together with the potent ability of Ba/F3-Y6 cells but not
Ba/F3-Fall cells to activate STAT5 (16), the involvement of STAT5 in
this cytokine-dependent NF-B activation was strongly suggested.
We next asked whether induction of NF-B activity may contribute to
expression of endogenous target genes in Ba/F3 cells. For this purpose,
we examined mRNA expression of proinflammatory cytokine IL-6, gene
expression of which is regulated by NF-B in various cell types and
known to be induced by IL-3 in Ba/F3 cells (54). Moreover,
analysis of the IL-6 gene seems appropriate for monitoring
NF-B-driven gene expression in the present study because the
promoter region of the IL-6 gene is lacking STAT5 response
element (55). As shown in Fig. 1E, the mRNA level of IL-6 was strongly induced within 3 h in response to IL-3 treatment in parental Ba/F3 cells. Furthermore, similar induction of IL-6 mRNA expression was observed after a 3-h stimulation of GM-CSF in
Ba/F3-wild and -Y6 cells. In contrast, treatment with GM-CSF did not
induce IL-6 mRNA expression in Ba/F3-Fall cells, reflecting the
incapability of this Fall mutant receptor to induce the activation of
NF-B. Taken together, these results clearly indicate that the
c-dependent NF-B activation leads to
expression of endogenous target genes including IL-6.
STAT5 Activates NF-B in Ba/F3 Cells--
Next,
we explored the possibility that STAT5 activation is involved in
cytokine-mediated NF-B activation. For this purpose, we
cotransfected into Ba/F3-wild cells an expression plasmid for WT STAT5A
together with the reporter plasmids for either NF-B or STAT5. As
shown in Fig. 2A, WT STAT5A
amplified GM-CSF-dependent induction of NF-B activity,
suggesting the primary role of STAT5 in NF-B activation in these
cells (left). The cotransfection efficiency of these
plasmids was confirmed by the enhancement of -casein promoter
activity induced by the presence of WT STAT5A in a
dose-dependent fashion (Fig. 2A,
right). The effect of STAT5B, an isoform of STAT5A, was also
examined, and the obtained results were quite similar to that of STAT5A
(data not shown).
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Fig. 2.
STAT5 activates NF-B
in Ba/F3 cells. Either 3 µg of NF-B-Luc (left) or
the -casein promoter-inducible luciferase reporter plasmid
-casein-Luc (right) were transfected into Ba/F3-wild
cells with the indicated amount of the expression vector
encoding murine WT STAT5A (A). After transfection, cells
were cultured for 12 h in the absence or presence of 5 ng/ml
GM-CSF. Luciferase activities were assayed and indicated as fold
activation as compared with those of cells transfected with empty
vector and left untreated. Either 3 µg of NF-B-Luc
(left top), 3 µg of -casein-Luc (right top),
or 10 µg of the AP-1 luciferase reporter plasmid AP-1-Luc
(bottom) were transfected into Ba/F3-wild cells with the
indicated amount of the expression vector encoding the constitutive
active mutant form of STAT5A (STAT5A1*6) (B). Another clone
of Ba/F3 cells stably expressing STAT5A1*6 (Ba/F3-STAT5A1*6) was also
transfected with the same amount of NF-B-Luc or -casein-Luc.
Cells were cultured in the depletion medium for 12 h and subjected
to assays for reporter activities. In the case of AP-1-Luc, cells
transfected with an empty vector were also cultured in
GM-CSF-containing medium. Luciferase activities were assayed and
indicated as fold activation as compared with those of cells
transfected with empty vector and left nonstimulated. Results are the
mean ± S.D. of 3 independent experiments.
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To bypass the effects of the other signaling cascades triggered
by the c rather than STAT5 activation, we also
analyzed the effect of a constitutive active mutant of STAT5,
STAT5A1*6, on NF-B activation in the absence of cytokine treatment.
STAT5A1*6 has two amino acid substitutions in the WT STAT5A backbone,
one in the DNA binding domain (H298R) and the other in the
transactivation domain (S710F) (18). This mutant is constitutively
phosphorylated on its tyrosine residue, localizes in the nucleus, and
is transcriptionally active in the absence of growth factor stimulation
(18). Efficient introduction of this strong transcriptional activator
was verified by the fact that -casein promoter activity was induced
in a dose-dependent manner of STAT5A1*6 even in the absence
of cytokine stimulation (Fig. 2B, top right). Of
note, overexpression of STAT5A1*6 significantly induced NF-B
activation despite the absence of cytokine stimulation (Fig.
2B, top left). We also investigated another
subline of Ba/F3 cells stably expressing STAT5A1*6 (Ba/F3-STAT5A1*6),
which are able to survive and proliferate without upstream cytokine
signals (18). As expected, Ba/F3-STAT5A1*6 cells exhibited strong
NF-B-dependent transcription in parallel with the
activation of STAT5-dependent reporter gene expression
(Fig. 2B, top). Analysis of AP-1-driven reporter
gene expression revealed that transcriptional activity of AP-1 was
induced by GM-CSF stimulation but not by STAT5A1*6 (Fig. 2B,
bottom). Taken together, we may conclude that STAT5 is
essential for the c-mediated NF-B activation in Ba/F3 cells.
STAT5 Enhances the DNA Binding Activity of NF-B without
Influencing Its Subcellular Localization--
To investigate the DNA
binding activity of NF-B and STAT5 in Ba/F3 cells, EMSA was
performed. As shown in Fig.
3A, two complexes were
observed between NF-B oligonucleotide probe and the nuclear extracts
prepared from GM-CSF-treated Ba/F3-wild cells (lane 1). These complexes were sequence-specific because not nonspecific (lane 5) but specific competitor DNA (lanes 2-4)
diminished the complex formation. Supershift assays revealed that
anti-p65 antibody shifted the upper band (lane 6) and
anti-p50 antibody shifted both upper and lower bands (lane
7). Antibodies against other Rel family proteins or Bcl-3, a
member of the IB family proteins, did not affect these complex
formations (lanes 8-11), indicating that the upper complex
is a heterodimer of p65/p50 and the lower complex is a p50/p50
homodimer. We obtained similar results when using the nuclear extracts
from Ba/F3 cells after IL-3 stimulation (Fig. 3B,
lanes 5, 10, and 15, and data not
shown). Note that the anti-STAT5 antibody, which recognizes both STAT5A
and STAT5B, could not supershift either of these NF-B complexes
(Fig. 3A, lane 12). We also demonstrated the
formation of protein-DNA complex between STAT5 oligonucleotide probe
and the nuclear extracts from GM-CSF-treated Ba/F3-wild cells (Fig.
3A, lanes 14-20).
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Fig. 3.
STAT5 activation downstream of the
c induces DNA binding activity of
NF-B. Ba/F3-wild cells were deprived of
IL-3 for 5 h and stimulated with 5 ng/ml GM-CSF (A).
After 3 h of stimulation, nuclear extracts were prepared and
subjected to EMSA with 32P-labeled oligonucleotide probes
containing NF-B (lanes 1-13) or STAT5 (lanes
14-20) binding sequences. Competition assays were
performed by adding different amounts of unlabeled specific competitor
(2-20-fold molar excess, lanes 2-4 and lanes
15-17) or nonspecific oligonucleotide spanning the glucocorticoid
response element (20-fold molar excess, lanes 5 and
18) in the reaction mixture. Supershift assays were
performed by preincubating the reaction mixture with either 2 µg of
the antibodies (Ab) against the indicated proteins
(lanes 7-12 and 19) or 2 µg of mouse IgG
(lanes 13 and 20) for 1 h prior to the
binding reaction. Arrows indicate the specific DNA-protein
complexes, and open arrows indicate the supershifted
complexes. Ba/F3-wild (lanes 1-5), -Y6 (lanes
5-10), and -Fall (lanes 11-15) cells were deprived of
cytokine for 5 h and restimulated with either 5 ng/ml GM-CSF or 4 ng/ml IL-3 (B). After the indicated time periods, nuclear
extracts were isolated and subjected to EMSA using the oligonucleotide
probes for STAT5 (top panels) and NF-B (bottom
panels).
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Based on these experimental settings, we examined the
time-dependent alteration of NF-B DNA binding in
response to GM-CSF stimulation in Ba/F3-wild, -Y6, and -Fall cells. As
shown in Fig. 4B, the DNA
binding activity of STAT5 was hardly detectable at the time point of
stimulation in any of these cells (top panels, lanes
1, 6, and 11), indicating the rapid
attenuation of STAT5 DNA binding activity after the removal of IL-3.
When stimulated with GM-CSF, the DNA binding activity of STAT5 reached
the maximum level within 15 min and was sustained for at least 3 h
in both Ba/F3-wild (lanes 1-4) and -Y6 (lanes
6-9) cells, whereas induction of STAT5 DNA binding was hardly
observed in Ba/F3-Fall cells (lanes 11-14). When assayed
for NF-B, all of these cells showed a substantial amount of NF-B
DNA binding activity in the nuclear extracts even after a 5-h
deprivation of IL-3 (bottom panels, lanes 1, 6, and 11). After GM-CSF stimulation, the DNA binding activity
of NF-B was induced ~2-2.5-fold at 1 and 3 h after
stimulation in Ba/F3-wild (bottom panel, lanes
1-4) and -Y6 cells (lanes 6-9). In Ba/F3-Fall cells,
however, the DNA binding activity of NF-B seemed weaker at 3 h
than that at the time point of stimulation (lanes 11-14). Treatment with IL-3 resulted in an increase in DNA binding of NF-B
in any of these cells (lanes 5, 10, and 15).
These observations indicate that cytokine receptor-mediated STAT5
activation enhances the DNA binding activity of NF-B.
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Fig. 4.
Nuclear translocation of
NF-B is not involved in
GM-CSF-dependent NF-B
activation. Ba/F3-wild cells were stimulated with 5 ng/ml
GM-CSF (left) or 20 ng/ml TNF (right) after
5 h of cytokine deprivation (A). After the indicated
time periods, cells were fixed and processed for immunocytochemical
analysis. Subcellular localization of p65 was analyzed by indirect
immunocytochemical staining using anti-p65 antibody as described under
"Experimental Procedures." FITC, subcellular
localization of p65 with fluorescein isothiocyanate; PI,
nuclear staining with propidium iodide; Merged, the merged
image of both. Ba/F3-wild cells were stimulated with GM-CSF
(left) or TNF (right) as described above
(B). After the indicated time periods, cells were harvested,
and both cytosol and nuclear extracts were isolated. Ten µg of each
was separated on a 10% SDS-polyacrylamide gel and immunoblotted with
anti-p65 antibody. Ba/F3-wild cells were stimulated with GM-CSF
(left) or TNF (right) and harvested at the
indicated time points, and whole cell extracts were prepared
(C). Ten µg of each was separated on a 10%
SDS-polyacrylamide gel and immunoblotted with anti-phosphorylated
IB (p-IB) antibody, and the protein blot was reprobed with
anti-IB antibody. As a control, whole cell extracts from cells
treated with TNF for 5 min were loaded on the same gel (lane
8). Immunoblotting with anti-IB antibody was also performed.
N.S., non-specific.
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Regulation of NF-B activity in most cell types involves the targeted
phosphorylation and degradation of IB proteins, allowing subsequent
nuclear translocation of NF-B (see the Introduction). To test this
in Ba/F3 cells, Ba/F3-wild cells were deprived of IL-3 for 5 h,
stimulated with GM-CSF, and processed for immunocytochemical staining
of p65 subunit of NF-B. As a control experiment, cells were treated
with TNF and examined in parallel. In TNF-treated cells, drastic
nuclear translocation of p65 was observed after 15-30 min of
stimulation (Fig. 4A, right, and data not shown). In contrast, GM-CSF-treated Ba/F3-wild cells exhibited no detectable nuclear translocation of p65 upon stimulation (Fig. 4A,
left). These striking differences were further confirmed by
Western blot analysis of NF-B p65. Ba/F3-wild cells were stimulated
with GM-CSF or TNF as above, and cytosol and nuclear extracts were
prepared. Fig. 4B shows that p65 proteins in the nuclear
extracts of TNF-treated cells were apparently increased after 15 min
of stimulation and then decreased within 180 min (Fig. 4B,
right). In clear contrast, p65 protein levels remained
almost constant even after GM-CSF stimulation (left).
Verification of our fractionation procedure was confirmed by Western
blot using anti-Sp1 antibody for nuclear extracts and
anti-c antibody of the GMR for the cytosol (data not
shown). Note that the presence of p65 protein in the nuclear extracts
even before cytokine stimulation is consistent with the previous
results showing substantial fluorescence signal of p65 staining (Fig.
4A) and the apparent DNA binding activity of NF-B (Fig.
3B). These results suggest that the
c-dependent NF-B activation is not
mediated through the classical activation pathway involving the
nuclear translocation of NF-B. This was further supported by the
following experiments in which the phosphorylation and degradation of
IB proteins were analyzed; both rapid phosphorylation of IB
and subsequent decrease of both IB and IB proteins were
detected in whole cell extracts from TNF-stimulated cells (Fig.
4C, right). However, phosphorylation of IB
was not observed, and the protein levels of both IB and IB
remained unchanged after GM-CSF stimulation (Fig. 4C,
left). There remains the possible mechanism that both
inducible but undetectable phosphorylation and rapid resynthesis of
IB proteins may occur, promoting the nuclear translocation of
NF-B, whereas the gross protein amount of IBs remains constant.
To test this, we also analyzed the time-dependent alteration of IB and IB in the presence of the protein
synthesis inhibitor cycloheximide in IL-3- or GM-CSF-treated Ba/F3-wild cells; however, the elimination of interference by newly synthesized IB proteins did not explore any degradative process of these proteins (data not shown).
These somewhat curious findings that the
c-dependent NF-B activation involves
enhancement of DNA binding activity without affecting its subcellular
localization prompted us to precisely investigate the effects of STAT5
on NF-B DNA binding activity. Previous reports described that when
Ba/F3 cells are deprived of cytokine, the DNA binding activity of
NF-B slowly declines (22). Thus, we also tested whether the decrease
in NF-B DNA binding following the cytokine deprivation was affected
by STAT5 activation. As shown in Fig.
5A, both STAT5 and NF-B are
recovered as DNA-bound complexes in both Ba/F3-wild and -Fall
cells cultured in the presence of IL-3 (top part,
lanes 1 and 5). The DNA binding activity of
NF-B was hardly discernible after a 9-h deprivation of IL-3
(lanes 4 and 8). Longer deprivation resulted in
total loss of the DNA binding, although cell viability was seriously impaired (data not shown). In the case of STAT5, deprivation of IL-3
rapidly decreased DNA binding in both of these cells (lanes 4 and 8 and data not shown). Interestingly, once
these cells were deprived of IL-3 but complemented with GM-CSF just
after IL-3 removal, the decline of DNA binding of STAT5 and NF-B was
inhibited in Ba/F3-wild cells (lane 3). This preservation of
NF-B DNA binding was of course observed after complementation with
IL-3 (lanes 2 and 6). In clear contrast, although
GM-CSF was complemented, preformed NF-B-DNA complexes in the absence
of IL-3 were not sustained in Ba/F3-Fall cells (lane 7),
suggesting the involvement of STAT5 in the preservation of the
c-dependent interaction between NF-B and
DNA. Because protein levels of p65 and p50 appeared to be constant
during the experiments (Fig. 5A, bottom part), it
was suggested that this alteration in NF-B DNA binding activity is
not due to a decrease of p65/p50 proteins. Moreover, subcellular localization of p65 in either of these cells was unaffected after a 9-h
deprivation of IL-3 as assessed by Western blot analysis (Fig.
5B), indicating that STAT5-dependent signals
downstream of the hGMR positively modulate the DNA binding activity of
NF-B without influencing either the subcellular sequestration or
protein amounts of p65/p50 complexes. To examine the effect of STAT5
more directly, we analyzed the effect of STAT5A1*6 on the DNA binding activity of NF-B. When the nuclear extract was prepared just after
electroporation of empty vector alone, the baseline NF-B DNA binding
was significantly detectable (Fig. 5C, bottom panel, lane 1). Relative loss of DNA binding of STAT5 in the same nuclear extract is reasonable because the cells had to stay cytokine-free for
nearly 1 h during the procedures of cytokine removal before electroporation (top panel, lane 1). The DNA binding of
NF-B then gradually decreased to the minimum level through 9 h
of culture in the depletion medium (lanes 1-4). In
contrast, when STAT5A1*6 was overexpressed, not only STAT5 but also
NF-B DNA binding was observed until 9 h after electroporation
even in the absence of IL-3 (top and bottom panels,
lanes 5-8). Thus, we may conclude that STAT5 is able to preserve
the DNA binding activity of NF-B within the nucleus. We next tested
the dose effect of STAT5 on the DNA binding activity of NF-B. For
this purpose, vector alone or indicated amounts of the expression
plasmid for WT STAT5A were transfected into Ba/F3-wild cells. Cells
were cultured in the presence or the absence of GM-CSF for 9 h,
and then EMSA was performed. The top panel of Fig.
5D represents that the DNA binding activity of STAT5
increased in proportion to increasing amounts of the expression
plasmids for WT STAT5A. In contrast, the DNA binding activity of
NF-B did not show a correspondent increase with the amounts of
transfected plasmids (bottom panel). Note that these doses
of the expression plasmids enhanced expression of NF-B-responsive reporter gene in a dose-dependent manner (Fig.
2A). These results, therefore, may argue the presence of an
additional mechanism that enhances the transcriptional function of
nuclear NF-B after DNA binding.
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Fig. 5.
Effects of STAT5 on constitutive DNA binding
of NF-B in Ba/F3 cells. A and
B, Ba/F3-wild and -Fall cells, grown in the culture medium
containing IL-3, extensively washed with the depletion medium, deprived
of IL-3, and then complemented with either IL-3 (lanes 2 and
6) or GM-CSF (lanes 3 and 7) or left
untreated (lanes 4 and 8). Cells were harvested
after 9 h of complementation as well as before substitution of
cytokines (lanes 1 and 5). Cells were collected,
and then cytosol and nuclear extracts or whole cell extracts were
isolated. Nuclear extracts were subjected to EMSA using the
oligonucleotide probes for STAT5 and NF-B (A, top part),
and whole cell extracts were separated on a 10% SDS-polyacrylamide gel
and immunoblotted with anti-p65 or anti-p50 antibody (A, bottom
part). Cytosol and nuclear extracts of Ba/F3-wild (B, top
panel) and -Fall (B, bottom panel) cells were also
separated on a 10% SDS-polyacrylamide gel and immunoblotted with
anti-p65 antibody. C, parental Ba/F3 cells, grown in the
culture medium containing IL-3 were deprived of IL-3 and transiently
transfected with 1 µg of either an empty vector (lanes
1-4) or the expression plasmid encoding STAT5A1*6 (lanes
5-8). After transfection, cells were cultured in the depletion
medium and harvested at the indicated time points. Nuclear extracts
were prepared and subjected to EMSA for STAT5 (top panel)
and NF-B (bottom panel). D, Ba/F3-wild cells,
grown in the culture medium containing IL-3, were deprived of IL-3 and
transiently transfected with either 1 µg of empty vector (lanes
1 and 2) or the indicated amounts of expression plasmid
encoding WT STAT5A (lanes 3-5). Cells transfected with
empty vector were cultured in the absence (lane 1) or
presence (lane 2) of 5 ng/ml GM-CSF, and those transfected
with WT STAT5A expression plasmids were cultured in the presence of
GM-CSF (lanes 3-5). After 9 h of culture, cells were
harvested, and nuclear extracts were prepared and subjected to EMSA for
STAT5 (top panel) and NF-B (bottom
panel).
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STAT5 Up-regulates Transactivational Function of p65--
To test
this hypothesis, we performed one hybrid assay to evaluate whether the
transactivational function of p65 is also affected by STAT5. We
constructed the indicated plasmids expressing GAL4 fusion proteins with
either the N- or C-terminal half of p65 (Fig. 6A). We then transfected each
of these plasmids, the GAL4-reporter plasmid, and either expression
plasmid for WT STAT5A or STAT5A749 into Ba/F3-wild cells, and cells
were treated with GM-CSF as indicated (Fig. 6B). As in the
case of GAL4 alone, the N-terminal half of p65 did not transactivate
reporter gene expression in the presence of either GM-CSF or WT STAT5A
(Fig. 6B). However, reporter gene expression in the presence of the
C-terminal half of p65 was significantly increased when WT STAT5A was
overexpressed (Fig. 6B), indicating that the
transactivational function of NF-B p65 is augmented by STAT5A.
Moreover, this effect of STAT5 was supposed to be ascribed to the
C-terminal transactivation domain because coexpression of STAT5A749
exhibited no detectable enhancement of reporter gene expression (Fig.
6B).
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Fig. 6.
STAT5 up-regulates transactivation potential
of NF-B p65. A, schematic
drawing of Gal4 fusion constructs. The indicated sequences of murine
p65 were fused to the DNA binding domain (DBD) of
Gal4 (amino acids 1-147), which is indicated as gray boxes.
B, Ba/F3-wild cells transiently transfected with 3 µg of
Gal4-inducible luciferase reporter plasmid in combination with 1 µg
of expression vectors encoding various Gal4 fusion proteins. The
indicated amounts of expression vectors encoding WT STAT5A or
STAT5A749 were also cotransfected. Cells were cultured in the
absence or presence of 5 ng/ml GM-CSF for 12 h and subjected to
luciferase assays. Luciferase activities were normalized to RLU per
µg of protein and indicated as fold activation as compared with those
of cells transfected with Gal4 alone and left nonstimulated. Results
are the mean ± S.D. of 3 independent experiments.
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Both DNA Binding Activity and Transactivational Functions of STAT5
Are Required for NF-B Activation--
To further elucidate the
mechanism for STAT5-dependent NF-B activation, we used
several mutants of STAT5 and performed cotransfection experiments.
STAT5AVVV mutant, in which amino acid residues Val-Val-Val (amino acids
466-468) within the DNA binding domain of STAT5A are replaced by
Ala-Ala-Ala, lacks DNA binding activity and is transcriptionally
inactive (42). STAT5A749, in which the C-terminal 44 amino acids
containing the transactivation domain are deleted, exerts
dominant-negative effects on wild-type STAT5-induced transcription despite retaining its DNA binding ability (56). As shown in Fig.
7, neither of these mutants was able to
augment NF-B activation in response to GM-CSF, suggesting that
NF-B activation requires both STAT5 DNA binding and transactivation
domains. These data raised the possibility that STAT5 induces
transcription of a certain gene, the product of which in turn enhances
NF-B function. We tried to confirm this possibility by using a
protein synthesis inhibitor cycloheximide on GM-CSF-induced NF-B
activation but failed because cycloheximide itself was a potent inducer
of NF-B DNA binding as estimated by EMSA in Ba/F3 cells (data not
shown). Therefore, we took another approach using STAT5750VP16JAK2,
which consists of the JAK2 kinase domain and mammary gland factor, the sheep homologue of STAT5, the transactivation domain of which is
replaced by that of VP16 (40). This chimeric protein constitutively activates STAT5-dependent transcription in the absence of
upstream cytokines (40). Interestingly, STAT5750VP16JAK2 could
induce NF-B activation even in the absence of cytokine stimulation
to the level comparable with that of STAT5A1*6 (Fig. 7). This
exchangeability of the transactivation domain of STAT5 may support the
indirect mechanism for NF-B activation, i.e. via
induction of as yet unidentified target gene expression.
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Fig. 7.
Both DNA binding and transactivation
functions of STAT5 are required for GM-CSF-dependent
NF-B activation. Ba/F3-wild cells were
transfected with 1 µg of an empty vector or either expression plasmid
for WT STAT5A or the indicated STAT5A mutant together with 3 µg of
NF-B-Luc. STAT5AVVV is a mutant in which three serial valine
residues in the DNA binding domain were converted to alanines (amino
acids 466-468). STAT5A749 is a mutant that lacks the C-terminal 44 amino acids containing the transactivation domain. STAT5750VP16JAK2
is a chimeric construct consisting of a JAK2 kinase domain (amino acids
757-1129) fused to the mammary gland factor whose transactivation
domain (amino acids 751-794) has been replaced by that of viral
protein VP16 (amino acids 411-489). Cells were cultured for 12 h
in the absence or presence of 5 ng/ml GM-CSF. Luciferase activities
were assayed and indicated as fold activation as compared with those of
cells transfected with empty vector and left nonstimulated. Results are
the mean ± S.D. of 3 independent experiments.
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STAT5-dependent Production of a Humoral Factor(s) Leads
to NF-B Activation in Ba/F3 Cells--
Cytokine-mediated STAT5
activation results in expression of several target proteins such as
Pim-1, CIS, and OSM (8, 19, 54). One recent report described
that OSM stimulated tissue factor expression in vascular smooth muscle
cells and that this transcriptional regulation is primarily mediated
through an IB-independent mechanism of NF-B activation (57).
Therefore, we hypothesized that STAT5-dependent gene
expression and secretion of certain soluble factors such as OSM may
participate in NF-B activation in Ba/F3 cells. To investigate such a
possibility, the culture supernatant of Ba/F3 cells was examined for
the capability to activate NF-B-dependent transcription.
We used the culture supernatant of Ba/F3-STAT5A1*6 cells as a
conditioned medium (CM STAT5A1*6) because these cells are able to
survive and proliferate in cytokine-deprived medium, and therefore, we
could eliminate the carry-over of IL-3, GM-CSF, and any factors
secreted via other signaling events downstream of the c.
Interestingly, as shown in Fig.
8A, CM STAT5A1*6 alone could
stimulate NF-B activity to a level comparable with that observed
after IL-3 stimulation in parental Ba/F3 cells (top). This
biological activity of CM STAT5A1*6 is supposed to be mediated through
a protein factor(s), because heat inactivation of CM STAT5A1*6 at
65 °C for 1 h nearly abolished the activity. Similar properties of inducing NF-B-dependent transcription were also
observed in the CM of several different clones of Ba/F3-STAT5A1*6 or
Ba/F3 cells in which another form of constitutive active mutant
STAT5A-N642H (58) is stably transfected (data not shown). An autocrine
mode of NF-B activation through secretion of IL-3 or GM-CSF was not likely because CM STAT5A1*6 induced neither STAT5- nor
AP-1-dependent transcription as determined by reporter gene
expressions (Fig. 8A, middle and bottom
panels). We next tested the possibilities of several other humoral
protein factors to be involved in this NF-B activation. As shown in
Fig. 8B, however, neither IL-6 nor OSM could induce NF-B
activation. In addition, IL-1, widely known as a potent inducer of
NF-B in multiple cell types, also had no effect (top).
TNF could stimulate NF-B-dependent reporter activities to a level comparable with that induced by IL-3 or CM
STAT5A1*6 (top); however, involvement of this cytokine in
STAT5-dependent NF-B activation seemed unlikely because
the mechanism of NF-B activation induced by TNF was distinct from
that induced by the c-mediated signals in Ba/F3 cells
(Fig. 5). In agreement with this, CM STAT5A1*6-dependent
NF-B activation was not blocked by the addition of neutralizing
antibodies against TNF (Fig. 8B, bottom), and
furthermore, enzyme-linked immunosorbent assay revealed no detectable
amount of TNF in the CM STAT5A1*6 (data not shown). Strikingly, we
found that this putative protein factor(s) in CM STAT5A1*6 could
activate not only reporter gene expression but also transcription of
endogenous NF-B target genes such as IL-6 (Fig.
8C). It should be emphasized that the
time-dependent increase of IL-6 mRNA in response to CM
STAT5A1*6 was earlier than that observed in the case of IL-3 or GM-CSF
stimulation (compare Fig. 8C with Fig. 1E). These
results strongly support our hypothesis that the
c-mediated NF-B activation is in some extent
dependent on a secondary mechanism involving
STAT5-dependent transcription and de novo
protein synthesis. In Western blot analysis, we again found that
phosphorylation and subsequent degradation of IB proteins are not
affected in this soluble factor-dependent NF-B
activation process (Fig. 8D). We, therefore, may conclude
that NF-B activation downstream of the c is mediated
at least in part through STAT5-dependent transcription and
the production of a(n) unknown humoral factor(s) that in turn evokes a
quite unique mode of NF-B activation.
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Fig. 8.
STAT5-inducible humoral factor activates
NF-B in Ba/F3 cells. Parental Ba/F3 cells
were transiently transfected with either 3 µg of NF-B-Luc
(top), 3 µg of -casein-Luc (middle), or 10 µg of AP-1-Luc (bottom) reporter plasmids (A).
Cells were then cultured for 12 h in either depletion medium alone
(MED), depletion medium plus 4 ng/ml IL-3 (MED + IL-3), conditioned medium obtained from parental Ba/F3 cells (CM
parent Ba/F3), or conditioned medium from Ba/F3 cells stably expressing
STAT5A1*6 (CM Ba/F3-STAT5A1*6). Heat inactivation of CM Ba/F3-STAT5A1*6
was performed at 65 °C for 1 h. Luciferase activities were
assayed and indicated as fold activation as compared with those of
cells cultured in the depletion medium. Parental Ba/F3 cells were
transiently transfected with 3 µg of NF-B-Luc and cultured in the
indicated conditions for 12 h (B). For cell
stimulation, 4 ng/ml IL-3 or various doses (5, 20, or 80 ng/ml) of
either IL-6, OSM, IL-1, or TNF was added to the depletion medium,
or cells were cultured in the CM Ba/F3-STAT5A1*6 (top). The
effects of anti-mouse TNF antibodies (Ab) were examined
by adding the indicated amounts of neutralizing antibodies or control
IgG (asterisks) 2 h prior to cellular stimulation with
20 ng/ml TNF or CM Ba/F3 STAT5A1*6 (bottom). Luciferase
activities were assayed and indicated as fold activation as compared
with those of cells cultured in the depletion medium. Parental Ba/F3
cells were deprived of IL-3 for 5 h, collected by centrifugation,
and then resuspended in CM Ba/F3-STAT5A1*6 (C and
D). Total RNA and whole cell extract were prepared after the
indicated time periods, and Northern blot analysis of IL-6 mRNA
(B) and immunoblot analysis of IB proteins
(D) were performed. N.S., non-specific.
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Humoral Factor(s) Produced by Ba/F3-STAT5A1*6 Could
Activate NF-B-dependent Transcription in WEHI 3B
Cells--
Finally, we examined whether STAT5 may induce NF-B
activation in different cell types other than Ba/F3 cells. When
myelomonocytic leukemic WEHI 3B cells were transiently transfected with
an expression vector encoding STAT5A1*6 along with reporter plasmid
-casein-Luc, significant induction of reporter gene expression was
observed (Fig. 9A,
left). In contrast, transfection of the same amount of
STAT5A1*6 expression plasmid did not augment but rather suppressed NF-B-dependent reporter gene activation
(right). These results indicate that the mechanisms by which
STAT5 up-regulates NF-
TOP
ABSTRACT
INTRODUCTION
