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INTRODUCTION |
Transforming growth factor
(TGF
)1 and its related
factors modulate essential cellular functions ranging from cellular
proliferation and differentiation to apoptosis (1-4). Signaling by
TGF
is initiated by an oligomeric receptor complex consisting of two types of transmembrane subunits that each possess serine/threonine kinase activity. Binding of ligand to the constitutively active type II
receptor (T
RII) promotes complex formation with the type I receptor
(T
RI/ALK5). Subsequent phosphorylation and activation of T
RI/ALK5
by T
RII leads to further propagation of TGF
signaling by several
signaling cascades, which include the Smads, MAPK, and PI3K (1-4).
Signaling by TGF
through the Smad pathway has been extensively
characterized and is considered the canonical pathway.
Receptor-regulated Smads (R-Smads), Smad2 and Smad3, are directly
phosphorylated and activated by ALK5. Phosphorylation occurs at
C-terminal SSXS motifs and promotes the formation of
heteromeric complexes with the common mediator Smad, or co-Smad, Smad4.
The Smad complexes translocate into the nucleus, where they regulate
gene expression by directly interacting with resident DNA-binding
proteins and by recruiting co-activators or co-repressors to the
promoter (1-4). Under basal conditions, R-Smads have been shown to be
retained in the cytoplasm through their interaction with
membrane-anchoring proteins containing FYVE domains, such as SARA (5)
and Hgs/Hrs (6), thereby facilitating R-Smad activation by TGF
receptors. Recently, we have shown that the adaptor molecule disabled-2
(Dab2) links TGF
receptors to Smad proteins (7), presumably in early endocytotic vesicles because of the interaction of Dab2 with the clathrin adaptor molecule AP-2 (8).
In addition to the canonical Smad pathway, TGF
has also been
reported to signal through components of the MAPK and PI3K/Akt pathways. TGF
has been shown to activate extracellular
signal-regulated kinase (ERK) (9, 10), Jun N-terminal kinase (JNK)
(11-14), p38 mitogen-activated protein kinase (p38) (15, 16), and
PI3K/AKT (17). The TGF
responses regulated by these kinases are
varied ranging from reporter construct transactivation to regulation of
cellular proliferation and apoptosis. The kinetics of these responses
also vary in magnitude and duration, and there are reports suggesting
that members of the Rho family of small GTPases may directly couple
activated TGF
receptors to these signaling pathways (11, 18-20) or
that activation of these pathways may be indirect, possibly resulting
from Smad-dependent transcriptional responses.
TGF
exerts both pro-apoptotic and anti-apoptotic effects depending
on the cell type or cellular context. Pro-apoptotic responses have been
demonstrated in prostate epithelium (21, 22), hepatocyte and hepatoma
cell lines (23-25), hematopoietic cells (26), and in B lymphocytes
(27-29). The molecular mechanisms mediating the pro-apoptotic effects
of TGF
are not completely understood and appear to be cell
type-dependent. Recently, it has been shown that Daxx, a
Fas-receptor-associated protein that activates the JNK pathway,
interacts directly with T
RII and couples TGF
signaling to the
apoptotic machinery in AML12 hepatocytes (30). In the Hep3B hepatoma
cell line, TGF
has been shown to induce Smad-dependent expression of the death-associated protein kinase (DAP-kinase), a
calcium/calmodulin-regulated serine/threonine kinase previously implicated in several apoptotic responses (31). Another study reports
that ARTS (apoptotic protein in the TGF
signaling pathway), a
septin-like protein, translocates from the mitochondria to the nucleus
in response to TGF
treatment of the prostatic epithelial cell line
NRP-154 (32). The Bcl-2 family of proteins has also been implicated as
mediators of TGF
-induced apoptosis. Early studies in the WEHI 231 B
lymphocyte cell line demonstrated that stable overexpression of
Bcl-XL abrogated TGF
-mediated apoptosis (27). More
recently, it was shown in the FaO rat hepatoma cell line that TGF
does not effect the expression levels of many members of the Bcl-2
family but did induce the caspase-dependent cleavage of
BAD, a pro-apoptotic Bcl-2 family member (33). Overexpression of Smad3
in these cells was shown to promote the caspase 3-mediated cleavage of
BAD and apoptosis, whereas antisense Smad3 cDNA blocked TGF
-mediated apoptosis and BAD cleavage (33).
We have recently shown that TGF
-mediated apoptosis in WEHI 231 B
lymphocytes can be blocked by overexpression of the inhibitory Smad7
protein (29). We further demonstrated that the transmembrane glycoprotein CD40, which has been shown to block or rescue B
lymphocytes from TGF
-induced apoptosis, can induce the expression of
the TGF
signaling inhibitor Smad7. Thus, the pro-survival signal transduction pathway(s) activated by CD40 induce the expression of
Smad7, which in turn, acts to down-regulate TGF
signaling (29).
In this study, we demonstrate that overexpression of the R-Smad, Smad3,
sensitizes WEHI 231 B lymphocytes to the apoptotic effects of TGF
.
We show that TGF
specifically induces the expression of the
pro-apoptotic protein Bim (Bcl-2-interacting
mediator of cell death), which is a BH3-only member of the
Bcl-2 family. Bim induction by TGF
is accompanied by increased
Bim/Bcl-2 heterodimerization and decreased mitochondrial membrane
potential. Furthermore, we find that CD40 activation abrogates the
TGF
-mediated induction of Bim. These results suggest that the
pro-apoptotic Bcl-2 family member Bim is a key mediator of the
apoptotic response in WEHI 231 cells and that its expression is
differentially regulated by either pro- or anti-apoptotic cytokines.
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MATERIALS AND METHODS |
Reagents--
TGF
2 was a generous gift from Genzyme Inc.
(Cambridge, MA) and was used at a final concentration of 5 ng/ml.
Purified hamster anti-mouse CD40 (
-CD40), rabbit anti-Bim antibody,
and mouse anti-Bad antibody were obtained from BD PharMingen (San
Diego, CA). Goat anti-mouse IgM (
-IgM) and mouse anti-Flag M2
antibodies, as well as reagent chemicals, were obtained from Sigma
Chemical Co. Protease inhibitor mixture tablets and the DNA molecular
weight standard (MWM XIV) were purchased from Roche Diagnostics
(Indianapolis, IN). Mouse anti-Bcl-2 (C-2), mouse anti-Bax (B-9),
rabbit anti-Bcl-XS/L (S-18), and rabbit anti-Hsp 90 (H-114)
antibodies and normal rabbit IgG were obtained from Santa Cruz
Biotechnology (Santa Cruz, CA). Rabbit anti-Smad3 antibody was from
Zymed Labs (San Francisco, CA). Secondary antibodies were purchased
from the following vendors: anti-mouse-IgG-HRP from Accurate Antibodies
(San Diego, CA) and anti-rabbit-IgG-HRP from Bio-Rad. Oligonucleotide
primers were obtained from Operon Technologies, Inc. (Alameda, CA).
DiOC6 (3) was purchased from Molecular Probes, Inc. (Eugene, OR).
Cell Culture and Transfection--
WEHI 231 cells were
maintained in T75 flasks at a density of 2 × 104
cells/ml in Dulbecco's modified Eagle's/F-12 medium supplemented with
5% fetal calf serum, 30 µM 2-
-mercaptoethanol, and
antibiotics (100 units/ml of penicillin and 100 mg/ml of streptomycin).
WEHI 231 clones that stably express FLAG-tagged-Smad3 or
FLAG-tagged-Smad3 dominant-negative (DN) proteins, as well as vector
controls, were produced by retroviral infection, as previously
described (29). The level of Smad3 or Smad3 DN expression was
determined by immunoblotting cell lysates with anti-Smad3 or anti-FLAG
M2 antibodies. Ba/F3 cells were maintained similar to WEHI 231 cells
except that conditioned medium from WEHI 3B cells was added to a final
concentration of 5% to provide the IL-3 essential for Ba/F3 survival.
Growth Inhibition Assay--
WEHI 231 cells (1 × 104 cells/ml) were cultured in T25 flasks at 37 °C in
the absence or presence of TGF
for up to 3 days. After each
treatment, cells were collected, and viable cells that excluded trypan
blue were counted.
Apoptosis Assays--
Apoptosis was demonstrated by DNA ladder
formation using either of the two following methods. Qualitative
assessment of DNA ladder formation was performed by isolating
oligonucleosomal DNA from cellular extracts and analyzing DNA by
ethidium bromide staining after electrophoresis through 2.0% agarose
gels, as described previously (29). Quantitative assessment of DNA
ladder formation was performed using the Cell Death Detection
ELISAplus kit (Roche Diagnostics, Indianapolis, IN).
Briefly, WEHI 231 cells (20 × 104 cells in 10 ml of
medium) were cultured in T25 flasks at 37 °C in the absence or
presence of TGF
for up to 48 h. Cells were collected at the end
of the experimental period and resuspended in 200 µl of kit lysis
buffer. The cellular lysate was centrifuged, and 20 µl of the
resulting supernatant was analyzed. Color development of the ELISA was
monitored spectrophotometrically at 405 nm. Results are expressed as
the ABS405 signal divided by the number of cells assayed. Apoptosis was
also demonstrated by TUNEL, as described previously (29).
RNA Preparation and Northern Analysis--
WEHI 231 cells
(4 × 106 cells in 40 ml of medium) were seeded into
T75 flasks and treated for up to 8 h with TGF
. The cells were
collected by centrifugation, and RNA was isolated using an RNeasy kit
from Qiagen (Valencia, CA). When 200 µg of RNA was accumulated from
several experiments, poly(A)+ RNA was isolated using an
Oligotex mRNA Mini kit from Qiagen and used for Northern analysis.
Northern analysis was carried out as described previously using 1%
formaldehyde-agarose gels (29). The Bim and
-actin cDNA probes
used in Northern analyses were obtained by RT-PCR using a Gene Amp PCR
Core kit from PerkinElmer Life Sciences (Roche Applied Science).
Briefly, 1 µg of total RNA from Smad 3D WEHI 231 cells was
reverse-transcribed using random primers. The cDNA template was
denatured at 94 °C, annealed at 48 °C, and extended at 72 °C
for 1 min each and amplified for 30 cycles to obtain three Bim-specific
PCR products of ~150, 250, and 450 bp. These three Bim PCR products
likely arise by amplification of the three major Bim mRNA isoforms,
BimEL, BimL, and BimS, as shown previously (34). The sequences of the
Bim primers were 5'-TCTGAGTGTGACAGAGAAGGTGGAC-3' for the forward primer
and 5'-CAGCTCCTGTGCAATCCGTATC-3' for the reverse primer. The cDNA
template was denatured at 94 °C, annealed at 60 °C and amplified
for 25 cycles to obtain a
-actin-specific 325-bp PCR product. The
sequences of the
-actin primers were 5'-CCAAGGCCAACCGCGAGAAGATGAC-3'
for the forward primer and 5'-AGGGTACATGGTGGTGCCGCCAGAC-3' for the
reverse primer. The PCR products were purified using a Wizard PCR Prep
column (Promega, Madison, WI) and 32P-labeled using a
Nick-translation kit (Roche Applied Science). Blots were hybridized
overnight at 42 °C in NorthernMax Hyb buffer (Ambion, Austin, TX)
and washed 3 × 20 min with 0.5× SSC, 0.1% SDS at 55 °C.
Quantitation of 32P-labeled probe hybridized to target
mRNA transcripts on Northern blots was accomplished using a
PhosphorImage analyzer (Molecular Dynamics, Sunnyvale, CA).
Nuclear Extract Preparation--
Nuclear and cytosolic extract
preparation for protein translocation experiments were performed as
described previously (29). Typically, 2-5 × 106
cells were resuspended 300 µl of hypotonic buffer (10 mM
HEPES, pH 8, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol plus protease
inhibitors), allowed to swell on ice for 15 min, and lysed by the
addition of 20 µl of 10% Nonidet P-40 with vortexing. The extract
was centrifuged at maximum speed for 1 min in a Beckman microfuge. The
resulting supernatant was termed the cytoplasmic extract. The pellet
was extracted in a high salt buffer (20 mM HEPES, pH 8, 25% glycerol, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol plus protease
inhibitors) for 20 min followed by centrifugation for 10 min. The
resulting supernatant was termed the nuclear extract. The protein
concentration of the extracts was determined using Bradford's reagent
(Pierce, Rockford, IL).
Mitochondrial Fractionation--
Mitochondrial and cytosolic
fractions were prepared from 1 × 107 WEHI 231 cells
using an ApoAlert cell fractionation kit from Clontech (Palo Alto, CA). Cells were treated in the
absence or presence of TGF
for 24 h and collected by
centrifugation. The cells were resuspended in 100 µl of ice-cold
fractionation buffer, incubated on ice for 30 min, and lysed by passing
50 times through a 26-gauge needle on a 0.5-ml syringe. The cytoplasmic
and mitochondrial fractions were prepared from the lysates following
the protocol supplied with the kit.
Western Blot Analysis--
Western blot analysis was performed
by standard SDS-PAGE, as described previously (29). Whole cell lysates
were prepared from 2-5 × 106 cells in 300 µl of
lysis buffer (20 mM Tris, pH 7.4, 1% Triton X-100, 10%
glycerol, 137 mM NaCl, 2 mM EDTA, 1 mM Na3V04, and protease
inhibitors). Lysates were sonicated and clarified by centrifugation at
4 °C for 10 min in a Beckman tabletop microcentrifuge at maximum
speed. Typically, 25-50 µg of whole cell lysates, 20 µg of nuclear
extracts, 50 µg of mitochondrial fractions, or 50-100 µg of
cytoplasmic extracts were separated on 10 or 12% acrylamide minigels
and transferred to Immobilon-P membrane (Millipore, Bedford, MA). The
membrane was blocked for 1 h in wash buffer (PBS containing 0.05%
Tween 20) containing 5% nonfat dry milk followed by a 2-h incubation
with primary antibody diluted in the same blocking buffer. After
extensive washing, the blot was incubated with secondary antibody for
1 h in blocking buffer, washed, and processed using the
ECL+ Western blotting detection system (Amersham
Biosciences). Primary antibodies were employed at a 1:500 to 1:2000
dilution, and secondary antibodies were used at a 1:1000 to 1:5000 dilution.
Co-Immunoprecipitation--
Whole cell lysates (500 µg) were
incubated overnight with 1 µg of either rabbit anti-Bim antibody or
normal rabbit IgG in 500 µl of whole cell lysate extraction buffer
containing protein G-agarose (Amersham Biosciences). The immune
complexes were collected by centrifugation and washed extensively with
whole cell lysate extraction buffer containing 500 mM NaCl.
The presence of Bcl-2 in the immune complexes was determined by Western blotting.
Mitochondrial Depolarization Assay--
Mitochondrial
depolarization was measured by FACS using the lipophilic cation
3,3'-dihexyloxacarbocyanine iodide, DiOC6 (3) (35). Typically, 2 × 106 cells were incubated for 24 h in the absence or
presence of TGF
, collected by centrifugation and resuspended in 300 µl of phosphate-buffered saline containing 1% bovine serum albumin.
DiOC6 (3) was added to a final concentration of 20 nM, and
the cells were stained for 15 min at 37 °C. Subsequently, the cells
were placed on ice without washing and subjected to FACS analysis. FACS
analyses were performed using a FACScan cytofluorometer (BD
Biosciences). Cells were gated to eliminate forward and side scatter.
DiOC6 (3) staining was monitored on 10,000 cells using the FL-1 channel.
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RESULTS |
Increased Smad3 Expression Sensitizes Cells to TGF
-induced
Apoptosis--
We have previously demonstrated that the inhibitory
Smad7 protein blocks TGF
-induced apoptosis in WEHI 231 B lymphocytes (29) suggesting a role for Smad proteins in mediating this signaling event. We sought therefore to more closely examine the role of the
R-Smad proteins, Smad2 and Smad3, in mediating the apoptotic effects of
TGF
in these cells. We retrovirally infected WEHI 231 cells to
overexpress FLAG-tagged Smad2 and Smad3, and single cell-derived clones
were selected in puromycin. While no Smad2-overexpressing clones could
be maintained, stable Smad3 overexpressing clones were obtained. Two
Smad3-overexpressing clones, designated S3D and S3E, were chosen for
further study. As shown in Fig.
1A, no endogenous Smad3 could
be detected by Western blotting of cellular lysates (100 µg of total
protein) from vector control cells. The Smad3-overexpressing clones
expressed different levels of Smad3, with S3D cells expressing higher
levels than S3E cells.

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Fig. 1.
Overexpressed Smad3 levels in WEHI 231 B
lymphocytes. A, basal Smad3 protein levels. Whole cell
lysates were prepared from vector control cells (Cont.) and
from two Smad3-overexpressing clones, termed Smad3D (S3D)
and Smad3E (S3E). The level of Smad3 protein (S3)
in 10-100 µg of whole cell lysates was determined by Western
blotting using an anti-Smad3 polyclonal antibody. B,
TGF -induced Smad3 phosphorylation. Vector control cells and Smad3D
cells were incubated in the absence (0) or presence of TGF for the
indicated times. At the end of the incubation nuclear fractions were
prepared and the level of phosphorylated Smad3 protein (S3P)
in 50 µg of nuclear fractions was determined by Western blotting
using a phosphospecific anti-Smad3 polyclonal antibody. C,
TGF -induced nuclear translocation of Smad3. Vector control, Smad3D,
and Smad3E cells were incubated in the absence ( ) or presence (+) of
TGF for 1 h. Nuclear and cytoplasmic fractions were prepared,
and the level of Smad3 protein (S3) was determined by
Western blotting using an anti-Smad3 polyclonal antibody.
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We next chose to determine the phosphorylation status and subcellular
localization of overexpressed Smad3 in the presence and absence of
TGF
. Fig. 1B demonstrates that both endogenous and
exogenous Smad3 are maximally phosphorylated in response to TGF
within 30-60 min and that phosphorylated Smad3 is present in the
nucleus. The results also demonstrate that endogenous Smad3, although
apparently expressed at very low levels in WEHI 231 cells, is readily
detectable in nuclear fractions using the phosphospecific Smad3
antibody (Fig. 1B). We next chose to determine the
subcellular localization of Smad3 in the overexpressing clones (S3D & S3E) under both basal and TGF
-stimulated conditions. As shown in
Fig. 1C, overexpressed Smad3 protein is present in both the
cytosolic and nuclear fractions under resting conditions. In the
presence of TGF
, additional Smad3 translocates to the nucleus with a
concomitant decrease in the cytoplasmic levels.
To determine the effect of Smad3 overexpression on TGF
-induced
apoptosis, we first examined cellular viability following TGF
treatment. As shown in Fig.
2A, TGF
stimulation results in a decrease in cellular viability with time. S3D cells, which express
the highest levels of Smad3 (Fig. 1A), show the greatest response to TGF
treatment, followed by the S3E and vector control transfectants. Also, as analyzed by ELISA (Fig. 2B) or by
oligonucleosomal DNA ladder formation (Fig. 2C), there was a
direct correlation between Smad3 expression levels and the extent of
TGF
-induced apoptosis. S3D cells, which express the highest levels
of exogenous Smad3 (Fig. 1A), showed the greatest
sensitivity to the apoptotic effects of TGF
.

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Fig. 2.
Increased Smad3 expression sensitizes cells
to TGF -induced growth arrest and
apoptosis. A, TGF -induced growth inhibition. Vector
control (Cont.), Smad3D (S3D), and Smad3E
(S3E) cells were seeded into T75 flasks (Day 0) and
incubated in the absence ( ) or presence (+) of TGF for up to 3 days without any change of the tissue culture medium. 1, 2, or 3 days
after seeding, the cells were collected and viable cell counts
determined. B, TGF -induced apoptosis. Vector control,
Smad3D, and Smad3E cells were incubated in the absence ( ) or presence
(+) of TGF for 24 h. A small aliquot of each sample was saved
for cell counts and the majority of the cells was used to quantitate
apoptosis by ELISA. Results are expressed as the absorbance reading
obtained from the ELISA normalized to the cell count. C,
TGF -induced DNA ladder formation. Vector control or Smad3D cells
were incubated in the absence ( ) or presence (+) of TGF overnight.
Oligonucleosomal DNA was isolated, electrophoresed through a 2%
agarose gel, and stained with ethidium bromide. A 100-bp DNA ladder
standard was run with the samples and is shown at the
left.
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TGF
Induces Bim Protein Expression--
Previous reports have
implicated members of the Bcl-2 family of proteins in mediating the
apoptotic effects of TGF
(27, 33). We therefore examined whether
TGF
-mediated apoptosis in the Smad3-overexpressing clones might be
associated with changes in the expression profile of Bcl-2 family
members. As shown in Fig. 3A,
immunoblot analysis of cellular lysates from Smad3-overexpressing S3D
cells revealed that TGF
treatment elicited its most significant effect on the pro-apoptotic family member Bim. While inhibitory effects
where observed on the expression levels of Bad, Bax, and Bcl-2
following a 24-h TGF
treatment, these could be secondary to the
apoptotic state of the cells. Bim expression levels, however, were
significantly increased as early as 2 h after TGF
addition and
typically continued to increase for at least 24 h in the presence of TGF
(Fig. 3A). Lysates were also tested for the
presence of Bcl-XS/L by immunoblot analysis, but no bands
were detectable. The levels of a cytosolic heat shock protein, Hsp-90,
were also analyzed and served as a protein-loading control.

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Fig. 3.
TGF induces Bim
protein expression. A, TGF -induced changes in the
protein level of Bcl-2 family members. Smad3D cells were incubated in
the absence (Cont.) or presence of TGF for up to 24 h. Whole cell lysates were prepared and 50-µg aliquots were analyzed
for the presence of Bim, Bad, Bax, Bcl-2, and Hsp-90 by Western
blotting. B, quantitation of TGF -induced increases in Bim
protein levels. Parental control (Cont.), Smad3D
(S3D), and Smad3E (S3E) cells were treated in the
absence or presence of TGF for 24 or 48 h. Whole cell lysates
were prepared and analyzed for the presence of Bim by Western blotting.
The high molecular weight isoform of Bim detected by Western blotting
was quantitated using Adobe Photoshop. Results show the fold-increase
over untreated controls. C, TGF induces apoptosis in
Ba/F3 cells. Wild-type Ba/F3 cells were incubated in the absence
(Cont.) or presence of TGF for up to 24 h. At the
times indicated oligonucleosomal DNA was isolated, electrophoresed
through 2% agarose gel, and stained with ethidium bromide. A 100-bp
DNA ladder standard was run with the samples and is shown at the
left. D, TGF induces Bim protein expression in
Ba/F3 cells. Ba/F3 cells were treated in the absence (Cont.)
or presence of TGF for the times indicated, and 50 µg of whole
cell lysates were analyzed for the expression of Bim and Hsp-90 by
Western blotting.
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The results shown in Fig. 3B demonstrate that while
TGF
induces Bim protein expression in the parental WEHI-231 cells,
the magnitude and kinetics of the induction were not the same as in the
Smad3-overexpressing S3D and S3E clones. TGF
-mediated Bim induction
in the S3D clone was on the order of 16-fold following a 48-h TGF
treatment, while in the parental WEHI-231 cells Bim induction was
~3-4-fold above non-stimulated levels (Fig. 3B). Also,
whereas TGF
-mediated induction of Bim protein was reliably observed
within 4 h in the S3D clone (Fig. 3A), its induction in
parental WEHI 231 cells was delayed and not observed until 24 h
after TGF
addition (data not shown). These results are consistent with the reduced TGF
-induced apoptosis observed in parental
versus Smad3 overexpressing cells, as shown in Fig. 2. The
results of Fig. 3B also demonstrate that induction of Bim
expression by TGF
occurs in two independent Smad3-overexpressing
clones (S3D and S3E), thus suggesting that the observed TGF
-mediated
induction of Bim in the S3D clone is not due to clonal selection. These results demonstrate that Smad3 overexpression results in a more rapid
and robust induction of Bim protein, which may be responsible for
potentiating the apoptotic effects of TGF
.
Previous studies have demonstrated increased Bim expression in the
mouse pre-B cell line, Ba/F3, during apoptosis induced by IL-3
withdrawal (36). We therefore wished to examine whether TGF
also
induced Bim expression and apoptosis in Ba/F3 cells. As shown in
Fig. 3, TGF
treatment of Ba/F3 cells resulted in a
time-dependent increase in oligonucleosomal DNA ladder
formation (Fig. 3C) and Bim protein expression (Fig.
3D). Following an 8 h TGF
treatment there was a
4.5-fold induction in Bim protein levels above control, non-stimulated
levels (Fig. 3D). Thus, in two progenitor B-lymphocyte cell
lines, WEHI-231 and Ba/F3, TGF
induces Bim protein expression,
providing further support for the crucial role of Bim in TGF
-induced
apoptosis in B lymphocytes.
Further support for the role of Smad3 in mediating Bim induction by
TGF
is provided by the results of Fig.
4A demonstrating the effects
of overexpression of dominant-negative forms of Smad3 on TGF
induction of Bim. In two distinct clones, S3.3DN and S3.8DN, TGF
-induced Bim protein expression is dramatically reduced
(lower panels) compared with the level of Bim induction by
TGF
in the Smad3-overexpressing clones, S3D and S3E (upper
panels). The expression level of dominant-negative Smad3 in the
S3.3DN and S3.8DN clones is shown in Fig. 4, panel B. The
results shown in Fig. 4C show that TGF
-mediated apoptosis
in these two clones, overexpressing dominant-negative Smad3, is reduced
relative to parental WEHI 231 cells, in agreement with our previous
study (29). Taken together, these results suggest that the induction of
Bim expression correlates with the TGF
apoptotic response in WEHI
231 cells and is potentiated by overexpression of Smad3.

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Fig. 4.
Smad3 dominant-negative abrogates
TGF -induced Bim protein expression.
A, effect of TGF on Bim protein levels in WEHI 231 clones
overexpressing Smad3 dominant-negative protein. Smad3D (S3D)
and Smad3E (S3E) cells as well as two independent WEHI 231 clones that overexpressed dominant-negative Smad3 (S3.3DN and S3.8DN)
were treated in the absence (Cont.) or presence of TGF
for 24 or 48 h. Whole cell lysates were prepared, and 50 µg were
analyzed by Western blotting for either Bim or Hsp-90. B,
quantitation of Smad3 dominant-negative protein levels. Whole cell
lysates were prepared from vector control (Cont.) cells and
the two Smad3 dominant-negative overexpressing clones (S3.8DN and
S3.3DN), as well as from COS cells transiently transfected with
dominant-negative Smad3 and analyzed by Western blotting for the
presence of exogenous dominant-negative Smad3 using an anti-Smad 3 antibody. C, TGF -induced apoptosis in Smad3
dominant-negative clones. Vector control cells (Cont.) as
well as S3.3DN and S3.8DN cells were treated in the absence or presence
of TGF for 24 h. Apoptosis was determined by the percentage of
TUNEL (+) cells, as described under "Materials and Methods."
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TGF
Induces Bim mRNA Expression--
To determine whether
the up-regulation of Bim expression by TGF
was a result of enhanced
transcription, Bim mRNA was analyzed (Fig.
5A). Bim has been shown to
have multiple isoforms generated by alternative splicing (34, 37, 38)
with three predominant isoforms, termed BimS, BimL, and BimEL for Bim
short (S), long (L), and extra long (EL). As depicted in Fig.
5A, we detected several Bim transcripts, in agreement with
previous reports (34), and expression of several of these mRNAs is
significantly elevated by TGF
addition. In particular, the largest
transcript (Fig. 5A, upper arrow) is induced
greater than 3-fold following a 4-8-h TGF
treatment (Fig.
5B). The lower, most prominent transcript (Fig.
5A, bottom arrow), did not show any significant
induction by TGF
. While Fig. 5A demonstrates that TGF
treatment induced the expression of several other less prominent
transcripts, these transcripts were not reliably observed by Northern
blot analysis.

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Fig. 5.
TGF induces Bim
mRNA expression. A, Northern analysis of Bim
mRNA levels. Smad3D cells were treated in the absence
(Cont.) or presence of TGF for up to 8 h.
Poly(A)+ RNA was isolated, and Bim mRNA transcripts
were detected by Northern analysis, as described under "Materials and
Methods." The blot was then stripped and analyzed for -actin
mRNA transcript levels by Northern analysis. The arrows
to the right of the Bim blot indicate the Bim mRNA
transcripts that were used for quantitation. B, quantitation
of Bim mRNA transcript levels. The amount of radioactivity in the
two Bim transcripts and in the -actin transcript of panel
A was quantitated by phosphorimage analysis. The amount of
radioactivity in the upper and lower Bim mRNA transcripts was
divided by the amount of radioactivity in the corresponding -actin
transcript. The ratio obtained for the untreated control
(Cont.) sample was set at 100%, and the ratios obtained for
the TGF -treated samples were normalized to this control value. The
results for the upper and lower Bim transcript levels are represented
by white and black bars, respectively.
C, effect of protein and mRNA synthesis inhibitors on
TGF -induced Bim protein expression. Smad3D cells were treated in the
absence ( ) or presence (+) of TGF for 8 h. During the last
4 h of TGF treatment, either cycloheximide (10 µg/ml) or
actinomycin D (0.5 µg/ml) was also present. Whole cell lysates were
prepared, and 50-µg aliquots analyzed for the presence of Bim, Bcl-2,
and Hsp-90 by Western blotting.
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In order to determine whether TGF
-induced Bim protein expression
required new protein synthesis or mRNA transcription, S3D cells
were co-incubated with cycloheximide or actinomycin D, respectively, during TGF
treatment. As shown in Fig. 5C, both
cycloheximide and actinomycin D treatment inhibited Bim protein
expression induced by TGF
. These two inhibitors had no effect on
either Bcl-2 or Hsp-90 protein levels, demonstrating that the
inhibition of Bim expression was not due to a nonspecific or toxic
effect. Taken together, the results of Fig. 5 are consistent with the
idea that TGF
-induced Bim expression occurs through a
transcriptional mechanism.
TGF
Promotes Mitochondrial Bim Accumulation, Bim
Heterodimerization with Bcl-2, and Loss of Mitochondrial Membrane
Potential--
Bim has previously been reported to translocate to the
mitochondria and form heterodimers with other Bcl-2 family members in
response to apoptotic stimuli (34, 39). It was of interest, therefore,
to determine the subcellular location of Bim induced by TGF
treatment. We performed immunoblot analysis on subcellular fractions of
S3D cells treated in the absence or presence of TGF
for 24 h.
As shown in Fig. 6A, TGF
induces the expression of Bim protein and has relatively little effect
on Bcl-2 expression levels in whole cell lysates (WCL). In
mitochondrial fractions, there is a significant increase in Bim protein
levels in the presence of TGF
and again little change in Bcl-2
levels in the absence or presence of TGF
. The results also
demonstrate that there is no significant amount of either Bim or Bcl-2
in the cytoplasm and that the two fractions are not cross-contaminated,
as indicated by the lack of the cytosolic protein Hsp-90 in the
mitochondrial fraction and the absence of the mitochondrial protein
Bcl-2 in the cytosolic fraction.

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Fig. 6.
TGF promotes
mitochondrial Bim accumulation, heterodimerization with Bcl-2, and loss
of mitochondrial membrane potential. A, subcellular
localization of Bim. Smad3D cells were treated in the absence ( ) or
presence (+) of TGF for 24 h. A small portion of the cells from
each sample was used to make a whole cell lysate whereas the majority
of the cell sample was used to prepare a mitochondrial and cytoplasmic
fraction. Aliquots of whole cell lysate (WCL, 50 µg),
mitochondrial fraction (Mito., 50 µg) and cytosolic
fraction (Cyto., 100 µg) were analyzed by Western blotting
for the presence of Bim, Bcl-2, and Hsp-90. B,
co-immunoprecipitation of Bim and Bcl-2. Smad3D cells were treated in
the absence (Cont.) or presence of TGF for up to 24 h, and whole cell lysates prepared. Bim protein was immunoprecipitated
from 500 µg of whole cell lysate and immunoprecipitates were analyzed
for the presence of Bcl-2 by Western blotting (upper blot).
Some lysates were immunoprecipitated with normal rabbit IgG as a
negative control. In addition, the whole cell lysates (50 µg) were
directly analyzed by Western blotting for the presence of Bcl-2
(middle blot) and Hsp-90 (lower blot).
C, mitochondrial depolarization induced by TGF . Smad3D
cells were incubated in the absence (Control) and presence
of TGF for 24 h. The cells were collected and stained with
DiOC6, as described under "Materials and Methods." The plot on the
right is an overlay of the two individual plots shown on the
left of fluorescence versus cell count for the
control and TGF samples.
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Since TGF
promotes accumulation of Bim in the mitochondria, we
wished to determine whether TGF
could also promote
heterodimerization of Bim with Bcl-2. The results of Fig. 6B
demonstrate by co-immunoprecipitation analysis that TGF
treatment
induces, in a time-dependent manner, complex formation
between Bim and Bcl-2. There is relatively little Bim associated with
Bcl-2 under basal conditions but TGF
stimulation promotes the
association of Bim with Bcl-2, with maximal effects observed between 8 and 24 h. Western blot analysis of the lysates used for
immunoprecipitation revealed a gradual, time-dependent reduction in Bcl-2 and Hsp-90 protein levels.
Bim protein has previously been shown to disrupt mitochondrial membrane
potential and promote the release of mitochondrial cytochrome
c, both early and critical events in many apoptotic processes (40). It was of interest, therefore, to determine whether
TGF
stimulation alters mitochondrial membrane potential in S3D
cells. We used the lipophilic cationic dye DiOC6 (3) to stain
mitochondria and monitored mitochondrial membrane depolarization by
FACScan analysis. The individual histograms shown to the left in Fig.
6C demonstrate that TGF
induces a decrease of DiOC6
staining following a 24-h treatment compared with untreated, control
cells. This depolarization is more apparent when the two histograms are overlaid, shown to the right of Fig. 6C. The M2 region of
the histograms represents healthy, propidium iodide-negative cells, whereas the M1 region represents damaged, propidium iodide-positive cells. Taken together, these results indicate that TGF
promotes an
increase in mitochondrial Bim protein levels, resulting in an increased
heterodimerization with Bcl-2 and a concomitant loss of mitochondrial
membrane potential.
CD40 Stimulation Antagonizes TGF
-mediated Apoptosis, Bim Protein
Expression, and Heterodimerization with Bcl-2--
The transmembrane
glycoprotein CD40 has been shown to couple to multiple signaling
pathways and its activation plays a critical role in promoting cellular
survival in numerous cell types, including WEHI 231 B lymphocytes (29).
In Fig. 7 we demonstrate by ELISA (Fig.
7A) and by oligonucleosomal DNA ladder formation (Fig.
7B) that activation of CD40 by
-CD40 antibody is able to
rescue or abrogate the apoptotic effects of TGF
in S3D cells. We
next determined whether CD40 rescue of TGF
-mediated apoptosis was
also associated with effects on Bim protein expression. The immunoblot
analysis shown in Fig. 7C demonstrates that co-stimulation
of cells with
-CD40 and TGF
inhibits the induction of Bim protein
mediated by TGF
. The induction of Bim protein following a 6- and
24-h TGF
treatment is inhibited to near basal levels following
co-stimulation with
-CD40. Furthermore, the results of Fig.
7D demonstrate that the TGF
-induced complex formation
between Bim and Bcl-2 following a 6- and 24-h TGF
treatment is
inhibited by
-CD40. These data demonstrate that CD40 stimulation can
rescue Smad3-overexpressing WEHI 231 cells from TGF
-mediated
apoptosis and suggest that inhibition of Bim protein induction by CD40
stimulation may represent a mechanism by which pro-survival cytokines
suppress the TGF
apoptotic response.

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Fig. 7.
CD40 antagonizes
TGF -mediated events. A, CD40
rescue of TGF -induced apoptosis. Smad3D cells were untreated
(Cont.) or treated for 24 h with TGF alone, CD40
alone or both TGF and CD40. A small aliquot of each sample was saved
for cell counts, and the majority of the cells was used to quantitate
apoptosis by ELISA. Results are expressed as the absorbance reading
obtained from the ELISA normalized to the cell count. B,
CD40 rescue of TGF -induced DNA ladder formation. Smad3D cells were
untreated (Cont.) or treated overnight with TGF alone,
CD40 alone, or both TGF and CD40. Oligonucleosomal DNA was isolated,
electrophoresed through a 2% agarose gel, and stained with ethidium
bromide. A 100-bp DNA ladder standard was run with the samples and is
shown at the left. C, CD40 blocks TGF -induced
Bim protein expression. Smad3D cells were untreated (Cont.)
or treated for 6 or 24 h with TGF alone, CD40 alone, or both
TGF and CD40. Whole cell lysates were prepared and 50-µg aliquots
were analyzed by Western blotting for the presence of Bim (upper
blot) or Hsp-90 (lower blot). D, CD40 blocks
TGF -induced Bim/Bcl-2 binding. Lysates (500 µg) used in
panel C, were immunoprecipitated with anti-Bim antibody or
normal rabbit IgG. The immunoprecipitates were analyzed by Western
blotting for the presence of Bcl-2.
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DISCUSSION |
Hematopoiesis is governed by a balance between opposing cell death
and survival programs that are, in turn, regulated by survival factors
and cytokines. It is well established that the Bcl-2 family, both pro-
and anti-apoptotic members, plays a crucial role in regulating these
programs (41, 42). Bim, the Bcl-2 interacting mediator of cell death,
is a recently discovered BH3-only member of the Bcl-2 family which is
expressed in hematopoietic tissues, as well as in epithelial, neuronal,
and germ cells (34, 43). Similar to other BH3-only family members, Bim
is thought to induce cell death by binding to and neutralizing
pro-survival Bcl-2 family members, thereby releasing Bax-like proteins
to execute cell death (44, 45). A specific role for Bim in mediating
hematopoietic cell death was demonstrated in lymphocytes isolated from
Bim knockout mice. Both B and T lymphocytes from Bim(
/
) mice
survive 10-30 times better than wild-type cells following cytokine
withdrawal, as well as after several other apoptotic stimuli (46, 47). The cell line used in our study, WEHI 231 cells, is an immature B-cell
line that is used extensively as a model of B cell tolerance and
apoptosis. Thus, these cells represent an attractive in
vitro model system to study the role of Bim in immunoregulation.
Here we show that TGF
induces the expression of Bim in WEHI 231 cells and that this induction is potentiated in Smad3-overexpressing cells.
Previous studies of BH3-only proteins have demonstrated that their
apoptotic function may be regulated by several different mechanisms
(48). The apoptotic function of Bad is regulated through
phosphorylation of two specific serine residues that abrogate its
binding to and neutralizing of the pro-survival proteins Bcl-2 or
Bcl-XL (49-51). Bid, however, is proteolytically cleaved
by active caspase 8, generating an active product termed tBid, for truncated Bid, that translocates to the mitochondria and induces apoptosis (52). Control of subcellular localization has also been
proposed as a regulatory mechanism for BH3-only proteins, including Bim
(39). Bim is sequestered to the microtubular motor complex by its
binding to dynein light chain (LC8) and following pro-apoptotic stimuli
is released into the cytoplasm allowing its interaction with
pro-survival Bcl-2 family members (39). More recent reports,
demonstrate that cytokine modulation of Bim expression levels represent
another mechanism of regulating the apoptotic function of BH3-only
proteins (36, 53). Specifically, IL-3 withdrawal from murine
hematopoietic progenitor cells results in an up-regulation of Bim
expression with an associated induction of apoptosis (36, 53). Similar
results are obtained when NGF is withdrawn from cultured neuronal cells
(45, 54, 55).
Our results presented here corroborate a model in which Bim expression
levels mediate cytokine regulated cell death. However, as opposed to
negative regulation of Bim expression levels by the pro-survival
cytokines IL-3 or NGF, we demonstrate that addition of a pro-apoptotic
cytokine, TGF
, results in the up-regulation of Bim expression levels
in two different B-cell lines, WEHI 231 and Ba/F3. This is the first
demonstration that addition, and not withdrawal, of a cytokine results
in enhanced Bim expression. We further demonstrate that the
pro-survival cytokine CD40 is capable of inhibiting the induction of
Bim expression in WEHI 231 cells in response to TGF
concomitant with
its rescue of the cells from TGF
-mediated apoptosis. Thus both pro-
and anti-apoptotic cytokines regulate Bim expression levels in WEHI 231 cells and underscore the pivotal role of this molecule in cytokine
regulation of cell survival and apoptosis.
Previous studies have implicated several signaling pathways as
mediating IL-3-induced repression of Bim expression levels in
hematopoietic cells, specifically Ba/F3 cells. Two Ras-activated pathways, involving Raf/MAPK and PI3K/mTOR, were shown to be
transducers of an IL-3-dependent down-regulation of Bim
expression concomitant with cell survival (53). Several reports have
also demonstrated that IL-2 and IL-3 regulate phosphorylation of the
forkhead family (FKHR) of transcriptional regulators in a
PI3K/AKT/PKB-dependent fashion to promote cell survival
through repression of Bim expression levels (36, 56). IL-3 was shown to
negatively regulate FKHR-L1 through phosphorylation of Thr-32 and
Ser-253 on FKHR-L1 correlating with a down-regulation of Bim expression
(36). Furthermore, inducible expression of exogenous FKHR-L1 resulted
in an elevation of Bim expression levels and induction of apoptosis,
suggesting that Bim expression is directly regulated by FKHR-L1 (36,
57).
In this study, we demonstrate that TGF
-mediated induction of Bim
expression is potentiated in Smad3-overexpressing WEHI 231 cells. While
it is well established that Smad proteins are key signaling components
in TGF
-mediated apoptosis, their precise role in regulating this
cellular process is still unclear (29, 33, 58-61). We have previously
shown that Smad7, the inhibitory Smad protein, abrogates TGF
-induced
apoptosis in WEHI 231 cells (29). Studies have also shown that
overexpression of wild-type Smad3 induces apoptosis in human lung
epithelial cells (58) and that overexpression of dominant-negative
forms of Smad3 inhibit TGF
induced apoptotic cell death in Hep3B
cells (63). More recently, TGF
-induced cell death in rat FAO cells
was shown to be potently enhanced by overexpression of Smad3 and
blocked by antisense Smad3 RNA expression (33). In this same report, it was shown that TGF
induced the cleavage of the BH3-only protein BAD
through an as yet to be determined Smad3-dependent
mechanism (33).
The findings presented here are noteworthy in that they identify the
pro-apoptotic protein Bim as a potential transcriptional target for
TGF
that is regulated in a Smad-dependent manner. Overexpression of Smad3 results in a sensitization of WEHI 231 B
lymphocytes to TGF
-mediated apoptosis (Fig. 1) concomitant with the
induction of Bim mRNA (Fig. 5) and enhanced expression of Bim
protein (Figs. 3, 4, and 6). Dominant-negative interfering forms of
Smad3 block TGF
-mediated cell death (29) and induction of Bim
protein expression (Fig. 4). The molecular mechanism through which
Smad3 may mediate Bim expression requires further investigation. It is
of note that members of the FKHR family of transcription factors were
the first Smad-interacting factors isolated and have subsequently been
demonstrated to serve as Smad transcriptional co-factors regulating, in
both a positive and negative manner, TGF
transcriptional responses
(64-66). Computer analysis of the 5' regulatory region of the murine
Bim gene (67) identified many putative Smad-DNA binding
elements (SBE), as well as consensus FKHR binding sequences. These data
suggest that combinatorial interactions between Smad3 and FKHR family
members could play a regulatory role in transcriptional activation of
the Bim promoter.
Our results also demonstrate that the pro-survival pathway induced by
CD40 results in abrogation of TGF
-mediated apoptosis and Bim
expression (Fig. 7). Interestingly, CD40 has previously been shown to
mediate its pro-survival effects in B lymphocytes through the PI3K/AKT
pathway (29, 68, 62), similar to that described for the IL-3 system. It
is not clear however, whether downstream of PI3K/AKT, CD40 regulates
FKHR transcriptional activation, as has been demonstrated for IL-3.
Thus, it appears that repression of Bim expression may be one of the
central mechanisms by which cytokines signaling through the PI3K/AKT
pathway mediate cell survival. It will be of interest to investigate
the potential role of forkhead transcription factors as a convergence
point between the TGF
/Smad3-mediated cell death pathway and the
CD40-mediated survival pathway in future studies.