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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 4 (February 15), 1999:
pp. 1346-1354
Both Stat3-Activation and Stat3-Independent BCL2 Downregulation Are
Important for Interleukin-6-Induced Apoptosis of 1A9-M Cells
By
Kenji Oritani,
Yoshiaki Tomiyama,
Paul W. Kincade,
Keisuke Aoyama,
Takafumi Yokota,
Itaru Matsumura,
Yuzuru Kanakura,
Koichi Nakajima,
Toshio Hirano, and
Yuji Matsuzawa
From The Second Department of Internal Medicine, The Department of
Hematology/Oncology, and The Department of Molecular Oncology,
Biomedical Research Center, Osaka University Medical School, Osaka,
Japan; and the Oklahoma Medical Research Foundation, Oklahoma City, OK.
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ABSTRACT |
A unique subclone of a bone marrow-derived stromal cell line,
BMS2.4, produces soluble factors that inhibit proliferation of several
types of hematopoietic cell lines. An understanding of these molecules
may be informative about negative regulatory circuits that can
potentially limit blood cell formation. We used expression cloning to
identify interleukin-6 (IL-6) as one factor that suppressed growth of a
pre-B-cell variant line, 1A9-M. Moreover, IL-6 induced
macrophage-differentiation and apoptosis of 1A9-M cells. During this
process, IL-6 downregulated expression of BCL2 in 1A9-M cells and
stimulated BCL-XL expression, but had no effect on p53, Bax, or Bak
gene expression. Mechanisms for transduction of IL-6-induced signals
were then evaluated in IL-6-stimulated 1A9-M cells. Whereas the signal
transducer and activator of transcription 3 (Stat3) was phosphorylated
and activated, there was no effect on either Stat1 or Stat5. The
importance of BCL2 and Stat3 on IL-6-induced
macrophage-differentiation and apoptosis was studied with 1A9-M cells
expressing human BCL2 or a dominant-negative form of Stat3,
respectively. IL-6-induced apoptosis, but not
macrophage-differentiation, was blocked by continuously expressed BCL2.
A dominant-negative form of Stat3 inhibited both
macrophage-differentiation and apoptosis induced by IL-6. However,
diminished Stat3 activity did not prevent IL-6-induced downregulation
of the BCL2 gene. Therefore, activation of Stat3 is essential for
IL-6-induced macrophage-differentiation and programmed cell death in
this model. Whereas overexpression of BCL2 abrogates the apoptotic
response, Stat3-independent signals appear to downregulate expression
of the BCL2 gene.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
BLOOD CELL FORMATION is strictly
regulated by the bone marrow microenvironmental factors, including
adhesion molecules, extracellular matrix components, and
cytokines.1-3 Interactions between very late antigen 4 and
vascular cell adhesion molecule 1 and between CD44 and hyaluronan play
an essential role, because antibodies against them blocked the
production of lymphoid and/or myeloid cells in long-term bone
marrow cultures.4-6 Modification of cell-cell adhesion by
an antibody against CD9 also blocked production of myeloid cells in
Dexter cultures.7 Regulatory cytokines in bone marrow are
typically produced in extremely small quantities, and some of them are
capable of binding to extracellular matrix.2
A number of genes that may be involved in lympho-hematopoiesis have
been identified by experiments using cloned stromal cell lines that
were originally selected for the ability to support proliferation
and/or differentiation of a particular type of hematopoietic cells. A stromal cell line, BMS2, which was established from adherent cells of long-term bone marrow cultures, has been shown to have the
capacity to support growth of pre-B cells.8 However,
BMS2.4, a subclone of BMS2, has unique characteristics. It blocked
proliferation of certain types of hematopoietic cells selectively and
interacted with the pH indicator, phenol red.9 We now
report that interleukin-6 (IL-6) is one of the growth-suppressors
produced by BMS2.4 cells. Recombinant human IL-6 as well as the
supernatant of cells transfected with mouse IL-6 cDNA induced
macrophage-differentiation and apoptosis of 1A9-M cells, a variant 1A9
pre-B-cell line.
IL-6 is a multifunctional cytokine that regulates proliferation and
differentiation of a variety of cells.10-12 IL-6 induces terminal differentiation of B cells to antibody-producing cells, proliferation of myeloma cells, and production of acute-phase proteins
by hepatocytes.13-15 IL-6 also induces macrophage
differentiation of several myeloid cell lines, including M1 and
Y6.16, 17 Recent studies have shown that IL-6 induced
phosphorylation and activation of signal transduction molecules, such
as Janus kinases (JAK1, JAK2, and Tyk2) and signal transducer and
activator of transcription family proteins (Stat1 and
Stat3).18,19 In particular, activation of the Stat3
molecule has been shown to be essential for IL-6-induced
macrophage-differentiation of M1 cells.20-22 We describe
here two IL-6-induced signal transduction pathways that lead to
macrophage-differentiation and apoptosis of 1A9-M cells.
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MATERIALS AND METHODS |
Cells.
A stromal cell line BMS2 and its subclone, BMS2.4, were cultured in
Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS; Flow Laboratories, North Ryde, Australia). 1A9, a pre-B-cell line, and its variant line, designated 1A9-M, were
cultured in McCoy's 5A medium supplemented with 5% FCS and 5 × 10 5 mol/L 2-mercaptoethanol.23 The 1A9-M
line has rearranged both JH alleles. However, one JH allele is
different from the original 1A9 line and therefore may have undergone a
secondary rearrangement. 1A9-M has also lost expression of IgM and
CD19. Lymphoma cell lines (7OZ/3 and EL4), an IL-3-dependent
pro-B-cell line (BaF3), and myeloid leukemia cell lines (FDCP1 and
WEHI3) were maintained as previously described.4
To make stable transformants of 1A9-M cells expressing human BCL2 or a
dominant-negative form of Stat3, pSVBT (a kind gift of Dr Y. Tsujimoto,
Osaka University, Osaka, Japan) or pCAGGS-Neo-HA-Stat3D21 were transfected into 1A9-M cells by electroporation and the
transformants were selected with 1 mg/mL of G418 (Sigma, St Louis, MO).
Expression cloning and sequence analysis.
Polyadenylated RNA was isolated from BMS2.4 cells using a Fast Track
mRNA isolation kit (Invitrogen, San Diego, CA). Double-stranded cDNA
was synthesized with a TimeSaver cDNA synthesis kit (Pharmacia, Uppsala, Sweden), ligated with BstXI adaptors (Invitrogen), and cloned into a mammalian expression vector, pEF-BOS (a kind gift of Dr
S. Nagata, Osaka Bioscience Institute, Osaka, Japan). Plasmid cDNAs
were purified from pools of a few hundreds of clones and were
transfected into 293T cells by calcium phosphate precipitation. Supernatants from each transfectant were added to cultures of 1A9-M
cells, and positive pools were identified by growth suppression of
1A9-M cells. Positive pools were divided into progressively smaller
pools and rescreened until single clones were isolated. The inserts of
the single clones were subcloned into pBluescript (Stratagene, La
Jolla, CA), and nucleotide sequence was determined using an automated
DNA sequencer (Applied Biosystems, Foster City, CA). Nucleotide data
base searching was performed with BLAST from the GCG computer program
(Genetics Computer Group, Madison, WI).
Flow cytometry.
Antibody incubations and washing steps were performed at
4°C in phosphate-buffered saline (PBS) containing 3%
heat-inactivated FCS and 0.1% sodium azide. Cells were analyzed with a
FACScan flow cytometer (Becton Dickinson, Mountain View, CA).
Antibodies used in this study were as follows: fluorescein
isothiocyanate (FITC)-conjugated antimouse CD32/CD16 (PharMingen, San
Diego, CA) and phycoerythrin (PE)-conjugated antimouse F4/80 (Caltag, San Francisco, CA). Isotype-matched Igs were used for controls.
Cell cycle analysis.
After stimulation, cells (1 × 106) were washed with
PBS, resuspended in 100 µL of PBS, and fixed by the addition of 900 µL of cold ethanol. The fixed cells were incubated with 300 µL of staining buffer (1 mg/mL RNase, 20 µg/mL propidium iodide, and 0.01%
NP-40 in PBS) at 37°C for 10 minutes. The DNA contents in the
nucleus of the cells were analyzed with FACSort (Becton Dickinson) using Cell Quest software.
DNA fragmentation assay.
Fragmentation of DNA was assayed as previously described.17
After stimulation with IL-6, 1A9-M cells (1 × 107)
were lysed in 0.4 mL lysis buffer containing 200 mmol/L Tris-HCl, 100 mmol/L EDTA, 1% sodium dodecyl sulfate (SDS), and 50 µg/mL proteinase K (Sigma) and incubated for 4 hours at 37°C. DNAs were extracted with phenol and then with chloroform/isoamylalcohol. The
aqueous phase was collected and precipitated with NaCl and ethanol. DNA
pellets were suspended in 0.4 mL TE buffer and treated with 50 µg/mL
RNase for 5 hours and then with 200 µg/mL proteinase K for 5 hours.
DNAs were extracted twice and precipitated as described above. DNA
pellets were resuspended in TE buffer, separated by electrophoresis in
1% agarose gel (1 µg DNA per lane), stained with 0.5 µg/mL
ethidium bromide, and visualized under UV light.
Luciferase assay.
A luciferase construct, 4× APRE-Luc, which contains a
Stat3-binding sequence, was used as a reporter gene.21,24
Luciferase assays were performed using the Dual-Luciferase Reporter
System (Promega, Madison, WI), in which transfection efficiency was
monitored by cotransfected pRL-CMV-Rluc, an expression vector for
renilla luciferase. The cultured cells were electroporated with 30 µg of reporter gene together with 30 µg of pRL-CMV-Rluc. The transfected cells were serum-starved for 12 hours and then stimulated with 20 ng/mL
IL-6 for 5 hours. The cells were lysed in lysis buffer supplied by the
manufactuer, followed by measurement of the firefly and the renilla
luciferase activities on luminometer LB96P (Berthold Japan, Tokyo,
Japan). The relative firefly luciferase activities were calculated by
normalizing transfection efficiency according to the renilla luciferase activities.
Northern blot analysis.
Total RNAs were isolated using TRIzol Reagent (GIBCO, Grand Island,
NY), electrophoresed through a formaldehyde agarose gel, and
transferred onto a nylon membrane (Amersham, Arlington Heights, IL).
The cDNA fragments were labeled with [32P]dCTP using a
random primed DNA labeling kit (Boehringer Mannheim, Indianapolis, IN)
and hybridized to the membrane. Blots were then washed and
autoradiographed. Fragments of the BCL2, BCL-XL, Bak, Bax, p53, and
 -actin genes were used as materials for probes.25
Western blot analysis.
The isolation of cellular lysates, gel electrophoresis, and
immunoblotting was performed according to the methods described previously, with minor modifications.7 Briefly, 1A9-M cells were lysed in lysis buffer, and insoluble material was removed by
centrifugation. Whole cellular lysates (15 µg per each lane) were
subjected to SDS-polyacrylamide gel electrophoresis. The proteins were
electrophoretically transferred onto a polyvinylidene difluoride
membrane (Immobilon; Millipore Corp, Bedford, MA). After blocking the
residual binding sites on the filter, immunoblotting was performed with
an appropriate antibody. Immunoreactive proteins were then visualized
with the enhanced chemiluminescence detection system (DuPont NEN,
Boston, MA). A mouse antihuman BCL2 antibody, a mouse antimouse BCL2
antibody, and a rabbit antimouse BCL-X antibody were purchased from
Santa Cruz Biotechnology, Inc (Santa Cruz, CA).
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RESULTS |
Supernatant of BMS2.4 suppressed growth of certain hematopoietic cell
lines.
BMS2.4 is an unique subclone of the BMS2 stromal cell line. In contrast
to the parent line that supports proliferation and differentiation of
lymphoid and myeloid precursors, BMS2.4 inhibits the growth of some
hematopoietic cell types.9 We evaluated whether these
inhibitory effects were mediated by soluble factors or cell surface
molecules. As shown in Fig 1, proliferation
of several hematopoietic cell lines (1A9, 7OZ/3, 1A9-M, and WEHI3) was
suppressed by BMS2.4 conditioned medium, whereas that of FDCP1, BaF3,
and EL4 cell lines was not affected. Supernatants derived from the
parent BMS2 line were not suppressive for any hematopoietic cell lines
tested. The 1A9-M cells appeared to be particularly sensitive to
inhibition by BMS2.4 and were used in subsequent attempts to identify
inhibitory factors. The growth-inhibitory activity was not blocked by
neutralizing antibodies to tumor necrosis factor- , tumor growth
factor-, or interferon- (data not shown).
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| Fig 1.
Growth inhibitory effects of BMS2.4 supernatant on
various hematopoietic cell lines. The indicated cell lines (1 × 104 cells/well) were cultured with supernatant of BMS2 or
BMS2.4 cells for 48 hours and number of remaining cells was determined
by hemocytometer counts. The results are shown as the mean ± SD of
triplicate cultures. Statistically significant differences from control
values are indicated: *P < .05; **P < .01.
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Identification of IL-6 as one inhibitory cytokine secreted by BMS2.4
cells.
Expression cloning of growth inhibitory substances was performed as
described in Materials and Methods. Approximately 1.2 × 105 clones of the BMS2.4 expression library were screened
on the basis of growth inhibition of 1A9-M cells, and 6 clones were
isolated (Table 1). Supernatants of 293T
cells that were transfected with plasmids prepared from these clones
showed more than 90% suppression of proliferation of 1A9-M cells.
Sequencing showed that all 6 clones contained mouse IL-6 cDNA inserts.
Moreover, proliferation of 1A9-M cells was suppressed in a
dose-dependent manner when recombinant human IL-6 was added to cultures
(Fig 2). Addition of a neutralizing
antibody to mouse IL-6 (Genzyme, Cambridge, MA) cancelled approximately
90% of the growth inhibitory effect of BMS2.4 supernatant on 1A9-M
cells (data not shown). Thus, IL-6 represents one major substance
produced by BMS2.4 cells that can arrest growth of 1A9-M cells.
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| Fig 2.
IL-6-induced growth inhibition of 1A9-M cells. (A) 1A9-M
cells were cultured with or without 20 ng/mL IL-6 for the indicated
time periods. (B) 1A9-M cells were cultured with various concentrations
of IL-6 for 3 days. The results of hemocytometer counts are shown as
the mean ± SD of triplicate cultures.
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IL-6 induced macrophage-differentiation and apoptosis of 1A9-M cells.
1A9-M cells typically have blastoid features with basophilic cytoplasm
and large nuclei containing nucleoli (Fig
3A, left panel) and are negative for CD45RA, Mac1 (CD18/11b), GR-1, and TER119 (data not shown). After treatment with IL-6, 1A9-M cells showed
macrophage-like morphologies, characterized by ample vacuolated cytoplasm and irregularly shaped nuclei (Fig 3A, right pannel). They
began to express Fc receptor (CD32/CD16) and a F4/80 antigen, a
macrophage-specific antigen, after IL-6 treatment (Fig 3B). 1A9-M cells
started to die within 48 hours of culture with IL-6 (Fig 4A). To clarify the mechanism of cell
death, nuclear DNA contents of 1A9-M cells cultured with IL-6 were
analyzed by flow cytometry. As shown in Fig 4B, the proportion of G0-G1
population increased within 24 hours (36.8% before stimulation; 46.4%
24 hours after stimulation), and the subdiploid peak appeared within 48 hours (0.2% before stimulation; 43.7% 48 hours after stimulation). Moreover, DNA obtained from 1A9-M cells cultured with IL-6 for more
than 48 hours showed extensive degradation with oligonucleosomal fragments (Fig 4C). Therefore, IL-6 induces macrophage-differentiation and apoptosis of 1A9-M cells. Whereas growth of a related pre-B-cell line, 1A9, was sensitive to factors produced by BMS2.4 (Fig 1), they
were not influenced in any obvious way by IL-6.
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| Fig 3.
IL-6-induced macrophage-differentiation of 1A9-M cells.
(A) 1A9-M cells were cultured with (right panel) or without (left
panel) 20 ng/mL IL-6 for 48 hours. The cells were prepared by cytospin,
stained by May-Grunwald-Giemsa, and photographed at 1,000×
magnification. (B) 1A9-M cells were cultured with or without 20 ng/mL
IL-6 for 24 hours. Surface expression of CD32/CD16 and a F4/80 antigen
was evaluated by flow cytometry.
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| Fig 4.
IL-6-induced apoptosis of 1A9-M cells. 1A9-M cells were
cultured with 20 ng/mL of IL-6 for the indicated time periods and then
subjected to (A) cell viability, (B) nuclear DNA content, and (C) DNA
fragmentation analysis as described in Materials and Methods. Each
figure shows one of three similar experiments.
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IL-6 modulated expression of apoptosis-related genes.
To investigate molecular mechanisms involved in 1A9-M cell responses to
IL-6, we evaluated expression of some apoptosis-related genes by
Northern blot analysis. As shown in Fig 5A,
1A9-M cells constitutively expressed BCL2 and Bax genes. Expression of
the BCL2 gene was downregulated within 24 hours after stimulation with
IL-6. In contrast, expression of the BCL-XL gene was induced by IL-6
treatment. Expression of Bax was not affected by IL-6 and neither the
Bak nor the p53 gene was expressed by these cells at a level detectable
by Northern blot. The BCL2 and BCL-XL proteins were also evaluated by
Western blot analysis. As shown in Fig 5B, 1A9-M cells lost BCL2
protein within 36 hours of stimulation with IL-6. Although expression
of BCL-XL mRNA was induced by IL-6 treatment, the protein was
undetectable even when 1A9-M cells were treated with IL-6 (data not
shown).
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| Fig 5.
(A) Expression of apoptosis-related genes during culture
with IL-6. 1A9-M cells were cultured with 20 ng/mL of IL-6 for the
indicated time periods. Total RNAs were isolated and subjected to
Northern blot analysis using the cDNAs of BCL2, BCL-XL, Bax, Bak, p53,
and -actin as probes. (B) Expression of BCL2 protein during culture
with IL-6. 1A9-M cells were cultured with 20 ng/mL of IL-6 for the
indicated periods. Whole cellular lysates were obtained and subjected
to Western blot analysis using an antimouse BCL2 antibody.
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Constitutive expression of BCL2 suppressed IL-6-induced apoptosis
but not macrophage-differentiation of 1A9-M cells.
Because IL-6 downregulated gene expression of BCL2, an antiapoptotic
gene, we evaluated the importance of this change for IL-6-induced
responses. A human BCL2-expression plasmid, pSVBT, was stably
transfected into 1A9-M cells, and 3 individual clones that expressed
human BCL2 under the control of a SV40 promoter (1A9-M/BCL2) were
obtained. The amount of gene and protein expression of endogenous and
exogenous BCL2 of 1A9-M/BCL2 clones was confirmed by Northern and
Western blot analysis (Fig 6A and B).
The1A9-M/BCL2 clones were cultured in the presence of IL-6 for 48 hours, and neither loss of viability nor apoptotic changes were
observed (Table 2). However, the 1A9-M/BCL2
clones did acquire the macrophage-like morphology, along with CD32/CD16
and F4/80 antigens after treatment with IL-6
(Table 3). These findings indicate that
programmed cell death is not coupled to differentiation in the 1A9-M
cell line and BCL2 is sufficient to block IL-6-induced apoptosis.
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| Fig 6.
Expression of BCL2 gene in 1A9-M/BCL2 clones and effects
of a dominant-negative form of Stat3 in 1A9-M/ST3D clones. (A) Total
RNAs from parent 1A9-M or 1A9-M/BCL2 clones were isolated and subjected
to Northern blot analysis using the cDNAs of BCL2 and -actin as
probes. (B) Whole cellular lysates were obtained from parent 1A9-M or
1A9-M/BCL2 clones and subjected to Western blot analysis using
antimouse BCL2 or antihuman BCL2 antibody. (C) Parent 1A9-M cells and
1A9-M/ST3D (clone 1, 2, A, B) were electroporated with 30 µg of a
reporter plasmid containing 4× APRE together with 30 µg of
pRL-CMV-Rluc. The cells were serum-starved for 12 hours and then
stimulated with 20 ng/mL IL-6 for 5 hours. The relative firefly
luciferase activities were calculated by normalizing transfection
efficiency according to the renilla luciferase activities. The results
are shown as the mean ± SD of triplicated experiments. ( )
Unstimulated; ( ) 20 ng/mL IL-6.
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Table 2.
Inhibition of IL-6-Induced Apoptosis of 1A9-M Cells
by a Dominant-Negative Form of Stat3 and by Constitutively Expressed
BCL2
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Table 3.
Inhibition of IL-6-Induced Macrophage
Differentiation of 1A9-M Cells by a Dominant-Negative Form of
Stat3, But Not by Constitutively Expressed BCL2
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Expression of a dominant-negative Stat3 blocked both IL-6-induced
apoptosis and macrophage-differentiation of 1A9-M cells.
IL-6 activates the JAK-STAT signal transduction pathway.18
Tyrosine phosphorylation of Stat3, but not Stat1 or Stat5, was observed
when 1A9-M cells were stimulated with IL-6 (data not shown). To
evaluate the importance of Stat3-mediated signals to IL-6-induced
macrophage-differentiation and apoptosis, we stably transfected a
pCAGGS-Neo-HA-Stat3D plasmid into 1A9-M cells. Twenty individual clones
were obtained that expressed a dominant-negative form of Stat3 carrying
mutations at positions important for DNA binding (1A9-M/ST3D). All
1A9-M/ST3D clones showed diminished levels of IL-6-activated
transcription from the 4× APRE reporter plasmid that contains a
potential Stat3-binding sequence (Fig 6C and data not shown). We then
examined biological responses of the 1A9-M/ST3D clones to IL-6. Neither
macrophage-differentiation nor apoptosis was induced by IL-6 when Stat3
functions were disrupted. Eighteen of 20 1A9-M/ST3D clones (clone 1-18)
did not undergo apoptosis in response to IL-6 (Table 2 and data not
shown). They also exhibit neither morphological changes nor acquisition
of macrophage antigens in response to IL-6 (Table 3 and data not shown). Thus, Stat3 was likely to mediate signals for IL-6-induced macrophage-differentiation and apoptosis of 1A9-M cells.
Interestingly, the other two clones (1A9-M/ST3D clones A and B) showed
unexpected responses to IL-6 (Tables 2 and 3). One clone (1A9-M/ST3D
clone A) had slight macrophage differentiation, although its viability
was retained. Approximately 30% of cells became apoptotic in another
stable clone (1A9-M/ST3D clone B), without differentiation into macrophages.
Stat3 was not involved in downregulation of BCL2 gene expression by
IL-6.
Experiments described above indicated that BCL2 might be a critical
determinant for life/death decisions in 1A9-M cells that are exposed to
IL-6. It seemed possible that the IL-6-induced downregulation of BCL2
gene expression might be dependent on Stat3. Therefore, we evaluated
BCL2 transcripts in the 1A9-M/ST3D clones before and after stimulation
with IL-6. As shown in Fig 7, IL-6 downregulated BCL2 gene expression within 24 hours in all of the 1A9-M/ST3D clones to an equivalent degree as in the control.
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| Fig 7.
A dominant-negative form of Stat3 did not inhibit
IL-6-induced repression of BCL2 mRNA expression. 1A9-M cells
transfected with either a control vector (1A9-M/pCAGGS clone 1 and 2)
or a pCAGGS-Neo-HA-Stat3D plasmid (1A9-M/ST3D clone 1 and 2) were
cultured with or without 20 ng/mL IL-6 for 24 hours. Total RNAs were
isolated and subjected to Northern blot analysis using cDNAs of BCL2
and -actin as probes.
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DISCUSSION |
BMS2.4 stromal cells probably produce multiple factors that modulate
the survival and growth of hematopoietic cells. Their identification
may be helpful in understanding cytokines that limit blood cell
production under steady state or conditions associated with unusual
demands. We used the particularly sensitive 1A9-M, a pre-B-cell line,
and expression cloning to determine that IL-6 is one of the active
substances produced by BMS2.4. The 1A9-M cells underwent remarkable
changes in morphology, expressed macrophage surface markers, and then
underwent apoptosis in response to IL-6. We demonstrated that these
dramatic responses are dependent on specific components of the JAK-STAT
pathway and that independent signaling mechanisms account for
downregulation of BCL2 expression.
Lymphocyte lines and normal IL-7-responding cells were unaffected by
IL-6, but inhibited by BMS2.4 supernatant. Therefore, the BMS2.4 line
must make other negative lymphopoietic regulators that can be
investigated using a similar experimental strategy. The 1A9-M cell line
was chosen for screening an expression library because of its
sensitivity to BMS2.4 supernatant inhibition. However, it is also
remarkable in its ability to undergo an apparent lineage switch before
programmed cell death. There have been several previous examples of
pre-B cells that have undergone a macrophage-like change. In some
cases, the change was spontaneous (HATFL and 7OZ/3), in others it was
induced by a proto-oncogene or ectopic expression of the c-fms
macrophage colony-stimulating factor receptor.26-32 This
may indicate that commitment between these two cell lineages involves
relatively few transcription factors. Regardless, we are unaware of
another cell line that undergoes this change before programmed cell
death. Whereas two myeloid cell lines, M1 and Y6, were previously found
to undergo macrophage-differentiation in response to IL-6, 1A9-M cells
may provide a unique model for exploring IL-6-dependent mechanisms.
Stat family members were originally identified as transcription factors
responsible for interferon-- and interferon- -dependent gene
expression.33 All Stat molecules have an SH2 domain, which recognizes phosphotyrosine in a specific peptide motif, and are generally involved in cytokine signal transduction.18,19
For examples, Stat1 is known to be involved in innate
immunity.34,35 Stat4 is activated by IL-12 and is involved
in the development of natural killer and T-helper type 1 cells.36,37 Stat6 is involved in Ig E production and
lymphocyte proliferation in response to IL-4.38,39 The
JAK-STAT pathway also mediates signal transduction through the IL-6
receptor subunit, gp130. JAK1, JAK2, and Tyk2 constitutively associate
with gp130 and respond to IL-6.40 These activated tyrosine
kinases, in turn, phosphorylate and activate the Stat family proteins,
especially Stat1 and Stat3 for IL-6.41,42 Recently, it was
reported that gp130 mutants without any of YXXQ motifs required for
Stat3 activation did not activate Stat3 or induce terminal
differentiation of M1 cells.20 Moreover, it was shown that
dominant-negative forms of Stat3 inhibited IL-6-induced macrophage-differentiation of M1 transformants.21,22
Although these findings indicate that Stat3 may be important for
IL-6-induced macrophage-differentiation, all of the experiments were
performed using the M1 cell line. We now report similar observations
with 1A9-M cells and can conclude that the phenomena may be general.
Apoptosis plays an important role in a wide variety of physiological
processes, including removal of redundant cells during development,
elimination of autoreactive lymphocytes, and eradication of old and
differentiated cells in most adult tissues with self-renewal capacity.43-45 Apoptosis of monocytes and macrophages has
emerged as a central regulatory event in hematopoiesis and
inflammation, and inflammatory cytokines can promote or prevent their
apoptosis.46 A number of genes, such as Bax, Bak, p53,
BCL2, and BCL-XL, have been reported to control apoptosis of cells and
to alter their gene expression during differentiation.47-49
In 1A9-M cells, IL-6 induced both macrophage-differentiation and
apoptosis. Expression of p53 and Bak genes was not detected in 1A9-M
cells, and BCL-XL was induced by IL-6 treatment. In contrast,
expression of BCL2 was downregulated by IL-6 treatment. Because BCL2 is
an inhibitor of apoptosis, downregulation of BCL2 may be related to
IL-6-induced apoptosis of 1A9-M cells. Indeed, downregulation of gene
and protein expression of BCL2 may be important for apoptosis, because
constitutively expressed BCL2 blocked IL-6-induced programmed cell
death. The cAMP response-binding proteins, p53, and c-myb are known to
regulate BCL2 gene expression positively or negatively in a variety of cells.50-52 In a pro-B-cell line, the tyrosine residue in
the YXXQ motifs of gp130 that is essential for Stat3 activation is
required for BCL2 induction and antiapoptotic effects.53
However, in 1A9-M cells, Stat3 was not related to regulation of BCL2,
because a dominant-negative form of Stat3 did not block IL-6-induced
downregulation of BCL2 expression. On the other hand, a Stat3-dependent
pathway is also required for IL-6-induced apoptosis of 1A9-M cells,
because a dominant-negative form of Stat3 maintained their viability. We could speculate that there is induction of apoptosis-related genes
other than p53, Bak, and Bax or reduction of some antiapoptotic genes
beside BCL2 and BCL-XL. In M1 cells, Stat3 is involved in IL-6-induction of several genes, such as the junB, interferon regulatory factor-1, a CDK inhibitor p19INK4D, and Stat3
itself, and in repression of c-myb and c-myc that may regulate
macrophage-differentiation and G1 growth arrest.21,54-56 It
will be interesting to analyze molecular mechanisms through which the
Stat3-dependent signalings induce apoptosis in 1A9-M cells.
We created stable 1A9-M transfectants using a dominant-negative form of
Stat3 and found that they have three patterns of response to IL-6.
Eighteen of 20 1A9-M/ST3D did not differentiate to macrophages or
undergo apoptosis in response to IL-6. The 1A9-M/ST3D clone A
differentiated to macrophages without apoptosis. In contrast, the
1A9-M/ST3D clone B died by apoptosis without macrophage-differentiation when stimulated. These three groups of cells differ from untransfected 1A9-M cells that differentiated to macrophages and died in response to
IL-6. Because the mutated Stat3 interfered with DNA binding of
endogenous Stat3 in all 1A9-M/ST3D clones, a second mutation due to
prolonged selection might occur in the exceptional clones (clone A and
B). These cells are suitable for substrates of subtraction techniques,
and further analysis may show the molecular mechanisms through which
IL-6 induces macrophage-differentiation and apoptosis.
| |
FOOTNOTES |
Submitted April 28, 1998; accepted October 9, 1998.
Supported in part by grants from the Ministry of Education, Science and
Culture, and the Japan Society for the Promotion of Science.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Kenji Oritani, MD, The Second Department of
Internal Medicine, Osaka University Medical School, 2-2 Yamada-oka,
Suita City, Osaka 565, Japan.
| |
REFERENCES |
1.
Long MW:
Blood cell cytoadhesion molecules.
Exp Hematol
20:288, 1992[Medline]
[Order article via Infotrieve]
2.
Kincade PW:
Cell adhesion mechanisms utilized for lymphohemopoiesis, in
Shimizu Y
(ed):
Lymphocyte Adhesion Molecules. Austin, TX, Landes, 1993, p 249.
3.
Verfaillie C, Hurley R, Bhatia R, McCarthy JB:
Role of bone marrow matrix in normal and abnormal hematopoiesis.
Crit Rev Oncol Hematol
16:201, 1994[Medline]
[Order article via Infotrieve]
4.
Miyake K, Medina K, Ishihara K, Kimoto M, Auerbach R, Kincade PW:
A VCAM-like adhesion molecule on murine bone marrow stromal cells mediates binding of lymphocyte precursors in culture.
J Cell Biol
114:557, 1991[Abstract/Free Full Text]
5.
Miyake K, Weissman IL, Greengerger JS, Kincade PW:
Evidence for a role of the integrin VLA-4 in lympho-hemopoiesis.
J Exp Med
173:599, 1991[Abstract/Free Full Text]
6.
Miyake K, Medina K, Hayashi S-I, Ono S, Hamaoka T, Kincade PW:
Monoclonal antibodies to Pgp-1/CD44 block lympho-hemopoiesis in long-term bone marrow cultures.
J Exp Med
171:477, 1990[Abstract/Free Full Text]
7.
Oritani K, Wu X, Medina K, Hudson J, Miyake K, Gimble JM, Burstein SA, Kincade PW:
Antibody ligation of CD9 modifies production of myeloid cells in long-term cultures.
Blood
87:2252, 1996[Abstract/Free Full Text]
8.
Pietrangeli CE, Hayashi S-I, Kincade PW:
Stromal cell lines which support lymphocyte growth: Characterization, sensitivity to radiation and responsiveness to growth factors.
Eur J Immunol
18:863, 1988[Medline]
[Order article via Infotrieve]
9.
Kincade PW, Medina K, Pietrangeli CE, Hayashi S-I, Naemen AE:
Stromal cell lines which support lymphocyte growth II. Characteristics of a suppressive subclone.
Adv Exp Med Biol
292:227, 1991[Medline]
[Order article via Infotrieve]
10.
Van Snick J:
Interleukin-6: An overview.
Annu Rev Immunol
8:253, 1990[Medline]
[Order article via Infotrieve]
11.
Hirano T:
The biology of interleukin-6.
Chem Immunol
51:153, 1992[Medline]
[Order article via Infotrieve]
12.
Hirano T:
Interleukin 6 and its receptor: Ten years later.
Int Rev Immunol
16:249, 1998[Medline]
[Order article via Infotrieve]
13.
Muraguchi A, Hirano T, Tang B, Matsuda T, Horii Y, Nakajima K, Kishimoto T:
The essential role of B cell stimulatory factor-2 (BSF-2/IL-6) for the terminal differentiation of B cells.
J Exp Med
167:332, 1988[Abstract/Free Full Text]
14.
Kawano M, Hirano T, Matsuda T, Taga T, Horii Y, Iwato K, Asaoku H, Tang B, Tanabe O, Tanaka H, Kuramoto K, Okada N, Kishimoto T:
Autocrine generation and essential requirement of BSF-2/IL-6 for human multiple myelomas.
Nature
332:83, 1988[Medline]
[Order article via Infotrieve]
15.
Gauldie J, Richards C, Harnish D, Lansdrop P, Baumann H:
Interferon 2/B-cell stimulatory factor type 2 shares identity with monocyte-derived hepatocyte-stimulating factor and regulates the major acute phase protein response in liver cells.
Proc Natl Acad Sci USA
84:7251, 1987[Abstract/Free Full Text]
16.
Miyaura C, Onozaki K, Akiyama Y, Taniyama T, Hirano T, Kishimoto T, Suda T:
Recombinant human interleukin 6 (B cell stimulatory factor 2) is a potent inducer of differentiation of mouse myeloid leukemia cells (M1).
FEBS Lett
234:17, 1988[Medline]
[Order article via Infotrieve]
17.
Oritani K, Kaisho T, Nakajima K, Hirano T:
Retinoic acid inhibits interleukin 6-induced macrophage differentiation and apoptosis in a murine hematopoietic cell line, Y6.
Blood
80:2298, 1992[Abstract/Free Full Text]
18.
Hibi M, Nakajima K, Hirano T:
IL-6 cytokine family and signal transduction: A model of the cytokine system.
J Mol Med
74:1, 1996[Medline]
[Order article via Infotrieve]
19.
Ihle JN:
STATs: Signal transducers and activators of transcription.
Cell
84:331, 1996[Medline]
[Order article via Infotrieve]
20.
Yamanaka Y, Nakajima K, Fukada T, Hibi M, Hirano T:
Differentiation and growth arrest signals are generated through the cytoplasmic resion of gp130 that is essential for Stat3 activation.
EMBO J
15:1557, 1996[Medline]
[Order article via Infotrieve]
21.
Nakajima K, Yamanaka Y, Nakae K, Kojima H, Ichiba M, Kiuchi N, Kitaoka T, Fukuda T, Hibi M, Hirano T:
A central role for Stat3 in IL-6-induced regulation of growth and differentiation in M1 leukemia cells.
EMBO J
15:3651, 1996[Medline]
[Order article via Infotrieve]
22.
Minami M, Inoue M, Wei S, Takeda K, Matsumoto M, Kishimoto T, Akira S:
STAT3 activation is a critical step in gp130-mediated terminal differentiation and growth arrest of a myeloid cell line.
Proc Natl Acad Sci USA
93:3963, 1996[Abstract/Free Full Text]
23.
Ishihara K, Medina K, Hayashi S, Pietrangeli C, Namen AE, Miyake K, Kincade PW:
Stromal-cell and cytokine-dependent lymphocyte clones which span the pre-B- to B-cell transition.
Dev Immunol
1:149, 1991[Medline]
[Order article via Infotrieve]
24.
Nakajima K, Kusafuka T, Takeda T, Fujitani Y, Nakae K, Hirano T:
Identification of a novel interleukin-6 response element containing an ETS-binding site and a CRE-like site in the JunB promoter.
Mol Cel Biol
13:3027, 1993[Abstract/Free Full Text]
25.
Tsujimura T, Hashimoto K, Morii E, Tunio GM, Tsujino K, Kondo T, Kanakura Y, Kitamura Y:
Involvement of transcription factor encoded by the mouse mi locus (MITF) in apoptosis of cultured mast cells induced by removal of interleukin-3.
Am J Pathol
151:1043, 1997[Abstract]
26.
Davidson WF, Pierce JH, Rudikoff S, Morse HC:
Relationship between B cell and myeloid differentiation. Studies with a B lymphocyte progenitor line, HAFTL-1.
J Exp Med
168:389, 1988[Abstract/Free Full Text]
27.
Hara H, Sam M, Maki RA, Wu GE, Paige CJ:
Characterization of a 7OZ/3 pre-B cell derived macrophage clone. Differential expression of Hox family gene.
Int Immunol
2:691, 1990[Abstract/Free Full Text]
28.
Tanaka T, Wu GE, Paige CJ:
Characterization of the B cell-macrophage lineage transition in 7OZ/3 cells.
Eur J Immunol
24:1544, 1994[Medline]
[Order article via Infotrieve]
29.
Bretz JD, Chen SC, Redenius D, Chang HL, Esselman WJ, Schwartz RC:
Lineage switch macrophages can present antigen.
Dev Immunol
2:249, 1992[Medline]
[Order article via Infotrieve]
30.
Spencker T, Neumann D, Strasser A, Resch K, Martin M:
Lineage switch of a mouse pre-B cell line (SPGM-1) to macrophage-like cells after incubation with phorbol ester and calcium ionophore.
Biochem Biophys Res Commun
216:540, 1995[Medline]
[Order article via Infotrieve]
31.
Klinken SP, Alexander WS, Adams JM:
Hemopoietic lineage switch: v-raf oncogene converts Emu-myc transgenic B cells into macrophages.
Cell
53:857, 1988[Medline]
[Order article via Infotrieve]
32.
Borzillo GV, Ashmun RA, Sherr CJ:
Macrophage lineage switching of murine early pre-B lymphoid cells expressing transduced fms genes.
Mol Cell Biol
10:2703, 1990[Abstract/Free Full Text]
33.
Darnell JJ, Kerr IM, Stark GR:
Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins.
Science
264:1415, 1994[Abstract/Free Full Text]
34.
Meraz MA, White JM, Sheedhan KCF, Bach EA, Rodig SJ, Dighe AS, Kaplan DH, Riley JK, Gleenlund AC, Campbell D, Carver-Moore K, DuBois RN, Clark R, Aguet M, Schreiber RD:
Targeted disruption of the mouse Stat1 gene in mice reveals unexpected physiologic specificity in the JAK-STAT signaling pathway.
Cell
84:431, 1996[Medline]
[Order article via Infotrieve]
35.
Durbin JE, Hachenmiller R, Simon MC, Levy DE:
Targeted disruption of the mouse Stat1 gene results in compromised innate immunity to viral disease.
Cell
84:443, 1996[Medline]
[Order article via Infotrieve]
36.
Kaplan MH, Sun YL, Hoey T, Grusby MJ:
Impaired IL12 responses and enhanced development of Th2 cells in Stat4-deficient mice.
Nature
382:174, 1996[Medline]
[Order article via Infotrieve]
37.
Thierfelder WE, van Deursen JM, Yamamoto K, Tripp RA, Sarawar SR, Carson RT, Sangster MY, Vignali DAA, Doherty PC, Grosveld GC, Ihle JN:
Requirement for Stat4 in interleukin-12-mediated responses of natural killer and T cells.
Nature
382:171, 1996[Medline]
[Order article via Infotrieve]
38.
Shimoda K, van Deusen JM, Sangster MY, Sarwar SR, Carson RT, Tripp RA, Chu C, Qualle FW, Nosaka T, Vignali S, Doherty PC, Grosveld G, Paul W, Ihle JN:
Lack of IL-4-induced Th2 response and IgE class switching in mice with disrupted Stat6 gene.
Nature
380:630, 1996[Medline]
[Order article via Infotrieve]
39.
Takeda K, Tanaka T, Shi W, Matsumoto M, Minami M, Kashiwamura S, Nakanishi K, Yoshida N, Kishimoto T, Akira S:
Essential role of Stat6 in IL-4 signaling.
Nature
380:627, 1996[Medline]
[Order article via Infotrieve]
40.
Lutticken C, Wegenka UM, Yuan J, Buschmann J, Schindler C, Ziemiecki A, Harpar AG, Wilks AF, Yasukawa K, Taga T, Kishimoto T, Barbieri G, Pellegrini S, Sendtner M, Heinrich PC, Horn F:
Association of transcription factor APRF and protein kinase Jak1 with the interleukin-6 signal transducer gp130.
Science
263:89, 1994[Abstract/Free Full Text]
41.
Stahl N, Farrugella TJ, Boulton TG, Zhong Z, Darnell JE, Yancopoulas G:
Choice of STATs and other substrates specified by modular tyrosine-based motifs in cytokine receptors.
Science
267:1349, 1995[Abstract/Free Full Text]
42.
Fujitani Y, Nakajina K, Kojima H, Nakae K, Takeda T, Hirano T:
Transcriptional activation of the IL-6 response element in the jun B promoter is mediated by multiple Stat family proteins.
Biochem Biophys Res Commun
202:1181, 1994[Medline]
[Order article via Infotrieve]
43.
Ellis RE:
Mechanisms and functions of cell death.
Annu Rev Cell Biol
7:663, 1991
44.
Green DR, Bissonnette RP, Glynn JM, Shi Y:
Activation-induced apoptosis in lymphoid systems.
Semin Immunol
4:379, 1992[Medline]
[Order article via Infotrieve]
45.
Williams GT:
Programmed cell death: Apoptosis and oncogenesis.
Cell
65:1097, 1991[Medline]
[Order article via Infotrieve]
46.
Mangan DF, Wahl H:
Differential regulation of human monocyte programmed cell death (apoptosis) by chemotactic factors and pro-inflammatory cytokines.
J Immunol
147:3408, 1991[Abstract]
47.
Yang E, Zha J, Jockel J, Boise LH, Thompson CB, Korsmeyer SJ:
Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death.
Cell
80:285, 1995[Medline]
[Order article via Infotrieve]
48.
Benito A, Grillot D, Nunez G, Fernandez-Luna JL:
Regulation and function of Bcl-2 during differentiation-induced cell death in HL-60 promyelocytic cells.
Am J Pathol
146:481, 1995[Abstract]
49.
Sanz C, Benito A, Silva M, Albella B, Richard C, Segovia JC, Insunza A, Bueren JA, Fernandez-Luna JL:
The expression of Bcl-x is downregulated during differentiation of human hematopoietic progenitor cells along the granulocyte but not the monocyte/macrophage lineage.
Blood
89:3199, 1997[Abstract/Free Full Text]
50.
Wilson BE, Mochon E, Boxer LM:
Induction of bcl-2 expression by phosphorylated CREB proteins during B-cell activation and rescue from apoptosis.
Mol Cell Biol
16:5546, 1996[Abstract]
51.
Haldar S, Negrini M, Monne M, Sabbioni S, Croce CM:
Down-regulation of bcl-2 by p53 in breast cancer cells.
Cancer Res
54:2095, 1994[Abstract/Free Full Text]
52.
Salomoni P, Perrotti D, Martinez R, Franceschi C, Calabretta B:
Resistance to apoptosis in CTLL-2 cells constitutively expressing c-Myb is associated with induction of BCL-2 expression and Myb-dependent regulation of bcl-2 promoter activity.
Proc Natl Acad Sci USA
94:3296, 1997[Abstract/Free Full Text]
53.
Fukada T, Hibi M, Yamanaka Y, Tezuka M, Fujitani Y, Yamaguchi T, Nakajima K, Hirano T:
Two signals are necessary for cell proliferation induced by a cytokine receptor gp130: Involvement of STAT3 in anti-apoptosis.
Immunity
5:449, 1996[Medline]
[Order article via Infotrieve]
54.
Nakajima K, Matsuda T, Fujitani Y, Kojima H, Yamanaka Y, Nakae K, Takada T, Hirano T:
Signal transduction through IL-6 receptor: Involvement of multiple protein kinases, stat factors, and a novel H7-sensitive pathway.
Ann NY Acad Sci
762:55, 1995[Medline]
[Order article via Infotrieve]
55.
Narimatsu M, Nakajima K, Ichiba M, Hirano T:
Association of Stat3-dependent transcriptional activation of p19INK4D with IL-6-induced growth arrest.
Biochem Biophys Res Commun
238:764, 1997[Medline]
[Order article via Infotrieve]
56.
Ichiba M, Nakajima K, Yamanaka Y, Kiuchi N, Hirano T:
Autoregulation of the Stat3 gene through cooperation with a cAMP-responsive element binding protein.
J Biol Chem
273:6132, 1998[Abstract/Free Full Text]
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Abstract
Introduction