ª
Oncogene (2002) 21, 8186 – 8191
2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00
www.nature.com/onc
Stat3-dependent induction of BATF in M1 mouse myeloid leukemia cells
Takeshi Senga1, Takashi Iwamoto2, Sean E Humphrey4, Takashi Yokota3,
Elizabeth J Taparowsky4 and Michinari Hamaguchi*,1
1
Department of Molecular Pathogenesis, Nagoya University School of Medicine, 65 Tsurumai Showa Nagoya 466-8550, Japan;
Radioisotope Research Center Medical Division, Nagoya University School of Medicine, 65 Tsurumai Showa Nagoya 466-8550,
Japan; 3Division of Stem Cell Biology, Graduate School of Medical Science, Kanazawa University, 13-1 Takaramachi, Kanazawa,
Ishikawa 920-8640, Japan; 4Department of Biological Sciences, Purdue University, West Lafayette, Indiana, IN 47907-1392, USA
2
Stat3 mediates cellular responses associated with
proliferation, survival and differentiation, but the
mechanisms underlying the diverse effects of this
signaling molecule remain unknown. M1 mouse myeloid
leukemia cells arrest growth and differentiate into
macrophages following treatment with interleukin 6
(IL-6) or leukemia inhibitory factor (LIF), and recent
studies have shown that Stat3 plays a central role in this
process. Utilizing representational difference analysis, we
demonstrate that expression of the mouse BATF gene is
upregulated as an early response to IL-6/LIF stimulation
and Stat3 activation in this cell system. Immunoblots
using antibodies to BATF detected an increase in BATF
protein in response to LIF/IL-6 stimulation. BATF is a
member of the AP-1 family of basic leucine zipper
transcription factors and functions to inhibit the
transcriptional and biological functions of AP-1 activity
in mammalian cells. BATF forms complexes with c-Jun
in M1 cells and forced expression of BATF in the
absence of Stat3 signaling results in a reduced rate of
cellular growth. These results indicate that Stat3
mediates cellular growth by modulating AP-1 activity
through the induction of BATF.
Oncogene (2002) 21, 8186 – 8191. doi:10.1038/sj.onc.
1205918
Keywords: IL-6/LIF; gp130; Stat3; BATF; AP-1
Members of the interleukin-6 (IL-6) cytokine family,
which include leukemia inhibitory factor (LIF), ciliary
neurotrophic factor (CNTF), oncostatin M (OSM),
interleukin-11 (IL-11) and cardiotrophin-1 (CT-1)
regulate cell growth, survival and differentiation and
are involved in variety of biological responses,
including the immune response, inflammation, hematopoiesis and oncogenesis. These cytokines use gp130
as a common receptor subunit. The binding of ligands
*Correspondence: M Hamaguchi;
E-mail: [email protected]
Received 21 January 2002; revised 24 July 2002; accepted 26 July
2002
to gp130 activates the JAK/Stat signal transduction
pathway where Stat3 plays a central role in transmitting signals from the membrane to the nucleus (Hirano
et al., 2000).
An increasing number of candidate target genes of
Stat3 have been characterized and many of these genes
encode proteins that regulate cell cycle progression
and/or modulate the apoptotic response. The expression of positive cell cycle regulatory genes such as
cyclin D1/D2/D3/A, cdc25A, c-myc, pim-1, bcl-2 and
bcl-x are up-regulated by Stat3 activation (Fukada et
al., 1996, 1998; Bromberg et al., 1999; Shirogane et al.,
1999). In contrast, Stat3 has been shown to contribute
to cell cycle arrest by inducing the expression of cyclindependent kinase inhibitors such as p19INK4D and
p21CIP1 (Bellido et al., 1998; O’Farrell et al., 2000).
The cellular circumstances, as well as the precise
molecular mechanisms, underlying the diverse signaling
effects described for Stat3 have yet to be determined.
To identify the genes involved in signal transduction
via gp130, we have performed representational difference analysis (RDA) using M1 cells which differentiate
into macrophages upon Stat3 activation by LIF/IL-6
(Lisitsyn et al., 1993; Hubank et al., 1994). Recently,
we reported that the Ral guanine nucleotide dissociation stimulator, RalGDS, was induced in this system in
a manner dependent on Stat3 activation (Senga et al.,
2001). We have analysed an additional 85 differentially
represented DNA fragments and found that eight
corresponded to the mouse BATF cDNA. BATF is a
125 amino acid nuclear basic leucine zipper protein and
a member of the AP-1 family of transcription factors
(Dorsey et al., 1995). BATF forms DNA binding
heterodimers with Jun proteins and negatively regulates
AP-1 mediated transcription in vitro and in vivo
(Dorsey et al., 1995; Echlin et al., 2000; Williams et
al., 2001). While the physiological function of BATF is
still unknown, the fact that BATF is restricted to
hematopoietic tissues suggests a role for the protein in
myeloid and/or lymphoid lineage development. To
confirm that BATF mRNA is induced upon LIFstimulation, we examined expression by RNA blot
analysis. BATF mRNA was induced 1 h after LIFstimulation, peaked at 3 h and declined thereafter
(Figure 1a). Stimulation of M1 cells with IL-6 had the
same effect (Figure 1b). Transcription of JDP-1 and
BATF induction in M1 cells
T Senga et al
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Figure 1 (a) Induction of BATF mRNA. M1 cells were stimulated with 5 ng/ml of recombinant murine LIF (Strathmann Biotech) for the times indicated and mRNA was extracted. 0.5 mg of
mRNA was resolved on denaturing gels and transferred to nylon
membrane (Amersham Pharmacia Biotech). The membrane was
hybridized with 32P-labeled BATF and G3PDH DNA probes.
(b) M1 cells were stimulated with 50 ng/ml of recombinant human
IL-6 (Kirin Brewery Co. Ltd.) and analysed by RNA blot hybridization as in (a). (c) M1 cells were stimulated with 5 ng/ml of recombinant murine LIF for 1 h and induction of JDP-1 and JDP2 mRNA was analysed by RNA blot hybridization as in (a). (d)
Cells were stimulated with indicated cytokines for 3 h and induction of BATF mRNA was examined. 32D cells were cultured in
the presence of IL-3 and stimulated with G-CSF. ES cells were
starved of LIF for 12 h and stimulated with 5 ng/ml of LIF. (e)
Full-length mouse BATF fused to GST was used to immunize a
rabbit. Anti-BATF antibodies were purified using GST-BATF
coupled to a HiTrap NHS-activated HP column (Amersham
Pharmacia Biotech). COS7 cells were transfected with pcDNA3/
Myc-tagged BATF and protein extracts analysed by immunoblotting. (f) M1 cells were stimulated for the times indicated and cell
lysates were immunoblotted with anti-BATF antibodies
JDP-2 – genes that encode proteins functionally related
to BATF by their ability to bind c-Jun and inhibit AP-
1 activity (Aronheim et al., 1997) were not induced by
LIF-stimulation (Figure 1c). We also examined the
induction of BATF mRNA in the 32D mouse cell line
and mouse ES cells. While 32D cells differentiate upon
Stat3 activation by G-CSF in the absence of IL-3, ES
cells proliferate in response to cytokines that activate
Stat3. Induction of BATF mRNA was seen in 32D, but
not in ES cells (Figure 1d), suggesting that the BATF
mRNA induction is a feature of cells undergoing
growth arrest in response to Stat3 signaling. The
observed increase in BATF transcripts is reflected in
an increase in BATF protein. Using polyclonal
antibodies raised to mouse BATF and reactive with
exogenously expressed Myc-tagged BATF in COS7
cells (Figure 1e), we detected increased expression of
BATF in M1 cells 6h after LIF-stimulation which
returned to basal levels by 12 h (Figure 1f).
To investigate whether the induction of BATF
mRNA was dependent on Stat3 activation, we
analysed BATF transcripts in M1 cells expressing a
dominant-negative form of Stat3 (Y705F) or a JAK
inhibitor (JAB /SOCS1/SSI-1) (Masuhara et al., 1997).
When stimulated with LIF, both M1/Y705F and M1/
JAB cells showed much weaker phosphorylation of
tyrosine 705 of Stat3 than parental M1 cells (Figure
2a). Corresponding to the depressed levels of Tyr705
phosphorylation, the induction of BATF mRNA also
was reduced (Figure 2b). We also generated M1 cells
expressing Stat3 fused with the estrogen receptor (M1/
Stat3ER). In these cells, Stat3 is conditionally active
following stimulation with 4-hydroxytamoxifen (4HT)
(Matsuda et al., 1999). Stimulation of M1/Stat3ER
with 4HT induced BATF mRNA (Figure 2c).
Together, these data strongly suggest that the induction
of BATF mRNA is dependent on the activation of the
JAK/Stat3 signaling pathway.
In addition to the JAK/Stat3 pathway, LIF/IL-6
also activates the SHP-2/Grb2/MAP kinase pathway
through gp130 (Fukada et al., 1996; Takahashi-Tezuka
et al., 1998). Therefore, we examined the possibility
that the induction of BATF might require the activity
of MAP kinase. As shown in Figure 3a, MAP kinase
was activated in M1 cells upon LIF-stimulation and
the MEK inhibitor, PD98059, inhibited this activation.
Interestingly, PD98059 had no effect on the induction
of BATF mRNA (Figure 3b), suggesting that signaling
via SHP-2/Grb2/MAP kinase does not play a major
role in the up-regulation of BATF. M1 cells were
stimulated with LIF in the presence of cyclohexamide
(CHX) to examine whether BATF induction required
de novo protein synthesis. As shown in Figure 3c, CHX
treatment did not affect BATF mRNA accumulation,
suggesting that BATF is likely to be a direct
transcriptional target of Stat3.
To examine the biological impact of BATF protein
induction in M1 cells, a Myc-tagged BATF expression
vector was introduced into M1 cells to establish cell
lines (M1/BATF 1 and 2). As a control, a cell line
expressing the neomycin resistance gene alone was
produced (M1/neo). Immunoblot analysis revealed that
the level of Myc-BATF expressed in these cell lines was
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Figure 3 (a) M1 cells were treated with 50 mM PD98059 and stimulated with LIF for 1 h. Cell lysates were immunoblotted with
anti-phospho-MAPK antibody (apMAPK) (Cell Signaling) or
anti-MAPK antibody (aMAPK) (Santa Cruz Biotechnology). (b)
M1 cells were stimulated with LIF plus 50 mM PD98059 or (c)
LIF plus 10 ng/ml cyclohexamide (CHX) and induction of BATF
mRNA was examined by RNA blot hybridization
Figure 2 (a) M1 cells, M1/705F cells and M1/JAB cells were stimulated with LIF for 1 h and cell lysates were immunoblotted
with anti-phospho-Y705 Stat3 antibody (ap705YStat3) (New England Biolabs) or anti-Stat3 antibody (aStat3) (New England BioLabs). (b) M1 cells, M1/705F cells and M1/JAB cells were
stimulated with LIF for 1 h and induction of BATF mRNA
was analysed by RNA blot hybridization. (c) M1/Stat3ER cells
were stimulated with 4HT for 3 h and induction of BATF mRNA
was analysed by RNA blot hybridization
approximately three times greater than the level
induced by LIF stimulation (Figure 4a). The morphology of cells was indistinguishable from that of parental
M1 cells. To examine if forced expression of BATF
accelerated the differentiation of M1 cells, additional
genes whose expression is altered by LIF-induced
differentiation in this system (Selvakumaran et al.,
1992; Lord et al., 1993) were examined. Expression of
BATF did not change the repression of myc and myb
genes, nor the induction of cd11b gene by LIF (data
not shown). The growth kinetics of M1/BATF1 and 2
cells were analysed and the results showed that the
growth rates of these cells were reduced to about one
half of that of M1/neo control cells (Figure 4b). Cell
cycle analysis revealed that the number of cells in the
G1 phase of the cell cycle was increased in M1/BATF1
and 2 cells relative to controls (Figure 4c). Since BATF
is thought to inhibit AP-1 activity through its
interaction with Jun proteins (Echlin et al., 2000), we
investigated if introduction of exogenous c-Fos to M1/
Oncogene
BATF cells might increase cell growth. Indeed, when cFos was introduced to M1/BATF1 and 2 cells, growth
increased (Figure 4b).
Since it has been reported that BATF binds to c-Jun
in vitro (Echlin et al., 2000), we examined if this
intermolecular interaction was occurring in M1 cells.
The Myc-BATF expressed in M1/BATF1 cells was
immunoprecipitated with anti-Myc antibody and the
proteins resolved by SDS – PAGE were immunoblotted
with an anti-c-Jun antibody. Results show the presence
of significant amounts of c-Jun in the BATF complex
(Figure 4d). We also tested if endogenous BATF
interacted with c-Jun. M1 cells were stimulated with
LIF and BATF was immunoprecipitated with the antiBATF antibody. As shown in Figure 4e, immunoblot
analysis of the proteins co-precipitated with BATF
using the anti-c-Jun antibody detected the presence of
c-Jun in endogenous BATF complexes as well. These
results are consistent with a model (Echlin et al., 2000)
in which BATF reduces AP-1 activity by competing
with Fos for the available intracellular pool of Jun.
To investigate if the induction of BATF results in a
change to the overall levels of AP-1 activity in LIFtreated M1 cells, luciferase activity from an AP-1
reporter gene was measured over time. As shown in
Figure 4f, AP-1 activity increases immediately after
LIF-stimulation. This is likely due to the induction of
the Jun family proteins, c-Jun and JunB, which
increase rapidly and remain elevated for at least 48 h
(Lord et al., 1993; data not shown). Interestingly, in
parallel with the expression pattern of BATF, which
peaks between 3 and 6 h after LIF exposure (Figure
BATF induction in M1 cells
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(legend overleaf)
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Figure 4 (a) Myc-tagged BATF is expressed in M1 cells. Myc-tagged BATF in the PMX-neo vector was used to transfect
BOSC23 cells as described previously (Kitamura and Morikawa, 2000). Forty-eight hours later, the medium was collected and
was applied to M1 cells with 10 mg/ml of hexadimethrine bromide (Sigma) for infection. Forty-eight hours after infection, cells were
selected with G418 (800 mg/ml) and two clonal cell lines (M1/BATF1 and M1/BATF2) were established. Lysates of M1, M1/
BATF1, M1/BATF2 and M1/BATF1 treated with LIF for 3 h were immunoblotted with anti-BATF antibodies. (b) c-Fos in the
pMX-puro vector was used to infect M1/BATF1 or M1/BATF2 cells and colonies selected with 10 mg/ml of puromycin. The growth
of M1/neo, M1/BATF1, M1/BATF2, M1/BATF1/FOS and M1/BATF2/FOS was compared. 26105/ml of cells of each line were
plated on day 0 and cell counts determined on days 1 – 3. Results represent the average of three independent experiments. (c) Cell
cycle analysis of M1/neo, M1/BATF1 and M1/BATF2 cells. Cells were fixed in 75% ethanol/water, stained with propidium iodide
and analysed by flow cytometry. (d) Anti-Myc antibody was used with lysates from M1/neo and M1/BATF1 cells to immunoprecipitate Myc-tagged BATF and associated proteins. Immunoprecipitates were resolved by SDS – PAGE and immunoblotted with
anti-c-Jun antibody (ac-Jun) and anti-Myc antibody (aMyc). (e) M1 cells were stimulated with LIF for the times indicated and cell
extracts were prepared. Proteins immunoprecipitated from the lysates using anti-BATF antibodies (aBATF) were immunoblotted
with a c-Jun and a BATF. Lysates from M1 cells treated with LIF for 3 h were subjected to immunoprecipitation with pre-immune
serum (P.I) as a control. (f) 16107 M1 cells per 100 mm plate were transiently transfected with 10 mg AP-1 Luc and 1 mg pRL-0Luc using the QIAGEN SuperFect1 protocol. Twenty-four hours after transfection, cells were treated with 5 ng/ml LIF for the
indicated times. Cells were lysed in 160 ml passive lysis buffer and 40 ml of each used to assay for AP-1 Luc activity, normalized
using the pRL-0-Luc internal control. AP-1 activity at time zero (prior to LIF treatment) was set to 1. Each time point represents
the average of three separate transfections, except for the 10 h time point which was performed once. Error bars indicate the s.e.m.
1f), there is a transient and reproducible decrease in
AP-1 luciferase activity. These results support a role
for BATF in modulating AP-1 transcriptional activity
during these early stages of LIF-induced differentiation.
To assess the importance of BATF induction to M1
cell differentiation, we attempted to inhibit BATF
expression by introducing anti-sense BATF, but
achieved only a partial block of BATF protein
accumulation and no obvious biological effect (data
not shown). We then attempted to express BATFTAD, a construct in which BATF is converted into a
potent activator of AP-1 transcription by fusion with
the transcription activation domain (TAD) of VP16
(Echlin et al., 2000). No neo resistant M1 colonies were
obtained. One possibility is that the high level of AP-1
transactivation directed by the fusion protein accelerates M1 cell differentiation and does not permit
colony expansion. This conclusion is supported by
studies (Lord et al., 1993) where c-Fos overexpression
was shown to accelerate M1 cell differentiation in the
presence of LIF. Since AP-1 luciferase activity
continues to be high in M1 cells (at least up to 10 h
following stimulation) (Figure 4f), it is likely that
BATF functions transiently to repress the expression of
key AP-1 targets whose orderly accumulation is critical
to the temporal control of differentiation in this
system.
BATF is expressed predominantly in hematopoietic
tissues where Stat3 is important for cell lineage
specification. It has been reported that Stat3 is required
for B cell differentiation into antibody-forming cells in
response to the stimulation of both CD40 and gp130
(Hirano et al., 2000; Ohtani et al., 2000). Stat3 also
plays an essential role in granulocyte colony stimulating factor (G-CSF)-induced granulocyte differentiation
(Shimozaki et al., 1997). The induction of BATF was
observed during the growth arrest and differentiation
of 32D cells upon G-CSF stimulation. These results
suggest a possible role for BATF in hematopoietic
differentiation by modulating AP-1 activity.
We have shown that LIF/IL-6 induces BATF
mRNA and protein in a Stat3 dependent manner. To
our knowledge, this is the first report of BATF as an
early response gene of cytokine stimulation. In
addition, we show that BATF interacts with c-Jun in
M1 cells and that expression of BATF parallels
changes in the expression of an AP-1 reporter gene.
Collectively, our results suggest a model in which Stat3
activation contributes to the suppression of M1 cell
growth by modulating AP-1 transcription through the
induction of BATF.
Acknowledgments
This work was supported by grants-in-aid for COE
Research from the Ministry of Japan Society for promotion of Science and by PHS award CA78264 to EJ
Taparowsky.
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