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INTRODUCTION |
Transforming growth factor-
(TGF-
)1
family members elicit their
multifunctional effects through heteromeric complexes of type I and
type II serine/threonine kinase receptors (1-3). The activated type I
receptor then propagates the signal through transient interaction with,
and phosphorylation of, receptor-restricted Smads (4, 5). Whereas Smad2
and Smad3 act in the TGF-
and activin pathways (6-9), Smad1, Smad5,
and Smad8 act downstream of bone morphogenetic proteins (BMPs)
(10-16). Receptor-mediated phosphorylation, which occurs on two serine
residues in a Ser-Ser-X-Ser motif at the end of the C-domain
(16-18), relieves the inhibitory activity of the N-domain and induces
homo- and hetero-oligomerization with each other and with Smad4 (a
common mediator of TGF-
family signaling) (19-21). This
hetero-oligomeric Smad complex then translocates to the nucleus and
modulates transcription of TGF-
family target genes (22-24).
Although we previously reported that activin A induces the cell cycle
arrest in the G1 phase and apoptosis in mouse B cell
hybridoma HS-72 cells (25), we are not aware of any reports on the
BMP-induced growth arrest in B lineage cells. Furthermore, little is
known of the precise mechanism by which BMP exerts its negative growth
effect. We report here that BMP-2 induces the cell cycle arrest and
apoptosis in B lineage cells.
Smad6 and Smad7 function as inhibitors of TGF-
family signaling
(26-30). Smad7 forms stable interactions with the activated TGF-
type I receptor, thereby preventing the activation of
receptor-restricted Smads by competition for receptor binding. Smad7
has also been shown to bind to and inhibit signaling downstream of BMP
receptors (29). In contrast, human Smad6 specifically blocks BMP but
not activin signaling in mink lung epithelial cells (30). Smad6 has
also been shown to bind to BMP receptors, inhibit signaling downstream
of the receptors (26), and prevent formation of an active Smad4/Smad1
signaling complex by directly competing with Smad1 for binding to Smad4
(30). Thus, Smad7 may act as a general TGF-
family inhibitor, and
Smad6 may have a tendency to preferentially inhibit the signal of BMP.
However, Imamura et al. (26) reported that Smad6 partially
inhibits the TGF-
signal for phosphorylation of Smad2 in COS-7 cells
and suppresses the activation of p3TP-Lux by TGF-
stimulation in
mink cells. In addition, Nakayama et al. (31) reported that
Xenopus Smad6, which is 52% identical to mammalian Smad6,
partially blocks the activity of activin in a mesoderm induction assay.
We have reported that the ectopic expression of Smad7 can block the
activin A-induced cell cycle arrest in the G1 phase and apoptosis in mouse B cell hybridoma HS-72 cells (32). In the present
study, we found that BMP-2 also induces growth arrest and apoptosis in
these cells. Furthermore, we compared the effects of Smad6 and Smad7 on
activin- and BMP-2-induced responses. In addition, we investigated the
effects of Smad6 and Smad7 on the phosphorylation status of
pathway-restricted Smads in HS-72 cells.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
Establishment and characterization of mouse
hybridoma HS-72 cells and Smad7-overexpressing clones (HS-72SM1 and
HS-72SM2) were described previously (32, 33). Cells were cultured in Iscove's modified Dulbecco's medium (Life Technologies, Inc.) containing 10% heat-inactivated fetal calf serum, 100 µg/ml
streptomycin, and 100 units/ml penicillin.
Plasmid and Transfection--
Construction of the Smad6
expression plasmid pcDNA3-FLAG-Smad6 was reported previously (26).
HS-72 cells were transfected with plasmid by electroporation using an
Electroporator II (Invitrogen, San Diego, CA) at 200 V and 1000 microfarad and then selected by cultivation with G418 (1 mg/ml).
Single-cell clones were obtained by limiting dilution.
MTT Assay--
The cells (2 × 104 cells/well
in 96-well plates) were incubated with Iscove's modified Dulbecco's
medium containing 5% fetal calf serum and antibiotics in the presence
of various concentrations of activin A or hBMP-2 for 48 h and then
examined for cell viability by a calorimetric assay with MTT (Sigma)
(33). Absorbance was determined at a wavelength of 570 nm with
background subtraction at 620 nm.
Detection of Apoptotic Cells--
To detect apoptotic nuclei,
the cells (2 × 105) were suspended in a hypotonic
solution (3.4 mM sodium citrate, 0.1% Triton X-100, 0.1 mM EDTA, 1 mM Tris-HCl (pH 8.0)), stained with
5 µg/ml propidium iodide, and analyzed by a FACSCalibur (Becton
Dickinson Immunocytometry Systems, San Jose, CA) (34). In the DNA
fragmentation assay, the DNA was extracted from HS-72 cells (5 × 106) according to the method of Moore and Matiashewski
(35). The DNA was electrophoresed in a 2% agarose gel and stained with
ethidium bromide.
Immunoprecipitation and Immunoblot Analysis--
Anti-FLAG
monoclonal antibody (anti-FLAG M2) was purchased from Eastman Kodak Co.
Anti-retinoblastoma protein (Rb) monoclonal antibody (G3-245) was
purchased from PharMingen (San Diego, CA). Anti-p21CIP1/WAF1
monoclonal antibody (Ab-4) was purchased from Calbiochem-Novabiochem. Anti-mouse Smad2 monoclonal antibody was purchased from Transduction Laboratories (Lexington, KY). The antibody against phosphorylated Smad2
was raised after immunizing rabbits with the synthetic peptide (KKKSSMS) phosphorylated at the last two serine residues coupled to
keyhole limpet hemocyanin as previously reported (9, 36). This antibody
specifically recognizes Smad2 phosphorylated by activated type I
receptor (36). The antibody against phosphorylated Smad1/Smad5 was
raised against KKKNPISSVS phosphorylated at the last two serine
residues and specifically recognizes Smad1 and Smad5 phosphorylated by
an activated type I receptor (36).
For immunoprecipitation, the cells were lysed in lysis buffer (1%
Nonidet P-40, 50 mM Tris-HCl (pH 7.5), 150 mM
NaCl, 1 mM phenylmethylsulfonyl fluoride, and a protease
inhibitor mixture) (complete, EDTA-free; Roche Molecular Biochemicals).
The extracted protein (600 µg) was reacted with 1 µg of goat
anti-Smad1/Smad5 serum (N-18, Santa Cruz Biotechnology Inc., Santa
Cruz, CA) at 4 °C for 1 h and then incubated with 20 µl of
protein G-Sepharose beads (Amersham Pharmacia Biotech) on ice for
1 h with gentle agitation. Immunoprecipitates were washed four
times with lysis buffer and analyzed as described below. For
immunoblotting, the cells were lysed in 50 mM Tris-HCl (pH
6.8) and 2% SDS, boiled for 5 min, and then centrifuged at 12,000 × g. The extracted proteins (60 µg) in supernatants were
separated by polyacrylamide gels containing 0.1% SDS and then
electroblotted on polyvinylidine fluoride membranes (Millipore Corp.,
Bedford, MA). Immunodetection was performed using an ECL Western
blotting detection system (Amersham Pharmacia Biotech). Blots were
stained with Coomassie Brilliant Blue, and each lane was confirmed to
contain a similar amount of protein.
Northern Blot Analysis--
Total RNA was extracted from the
cells using an Isogen RNA extraction kit (Nippon Gene, Tokyo, Japan).
Total RNA (10 µg) was electrophoresed in a formaldehyde-agarose gel
and blotted on a nylon membrane (Hybond-N+, Amersham
Pharmacia Biotech). Mouse Smad6, mouse Smad7, mouse p21CIP1/WAF1, and glyceraldehyde-3-phosphate dehydrogenase
cDNA fragments were isolated from pcDNA3-FLAG-Smad6 (26),
pcDNA3-FLAG-Smad7 (28), pCMW35T3, and pKS321 (37), respectively,
and labeled with [
-32P]dCTP using a
MultiprimeTM DNA labeling system (Amersham Pharmacia
Biotech). Hybridization and washing were performed as described
previously (38).
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RESULTS |
Activin A and BMP-2 Induce Inhibitory Smad mRNA
Expression--
Northern blot analysis of Smad7 revealed that Smad7
mRNA was induced in HS-72 cells stimulated with either hBMP-2 or
activin A (Fig. 1A). The
expression of Smad7 mRNA was most prominently induced after 1 h of hBMP-2 or activin A stimulation and decreased time dependently.
Northern blot analysis of Smad6 revealed that Smad6 mRNA was
induced in HS-72 cells stimulated with either hBMP-2 or activin A (Fig.
1B). Maximal levels of Smad6 mRNA expression were
achieved after 3 h of stimulation with either activin A or hBMP-2.

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Fig. 1.
Activin A and hBMP-2 induce Smad6 and Smad7
mRNA in HS-72 cells. HS-72 cells were exposed to activin A (50 ng/ml) or hBMP-2 (50 ng/ml) for various times. The total RNA (10 µg)
was separated and analyzed for levels of Smad7 (A) and Smad6
(B) mRNA by Northern blotting. The same blot was
serially hybridized with a glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) cDNA probe.
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Effects of Inhibitory Smads on BMP-2- and Activin A-induced Growth
Arrest--
To investigate whether Smad6 controls the growth arrest
induced by activin A or hBMP-2, HS-72 cells were stably transfected with mouse Smad6 expression plasmid (pcDNA3-FLAG-Smad6). Two clones (HS-72Sma6A and HS-72Sma6B) were found to express high levels (66 kDa)
of mouse FLAG-Smad6 (Fig. 2A).
Immunoblot analysis showed that the expression levels of FLAG-Smad6 in
HS-72Sma6A and HS-72Sma6B were similar to those of FLAG-Smad7 in
HS-72SM1 and HS-72SM2.

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Fig. 2.
Effects of ectopic expression of Smad6 and
Smad7 on growth arrest in HS-72 cells. HS-72 cells were
transfected with plasmid pcDNA3-FLAG-Smad6. FLAG-Smad6 and
FLAG-Smad7 expressions were detected using an anti-FLAG monoclonal
antibody by immunoblotting (A). The cells were cultured with
various concentrations of hBMP-2 (B and C) or
activin A (D) for 48 h, and the cell viabilities were
monitored by MTT assay. Percent viability was calculated by the
following formula: percent viability = 100 × (A570-620 nm with
hBMP-2/A570-620 nm without
hBMP-2). Closed triangle, HS-72C4; open triangle,
HS-72SM1; ×, HS-72SM2; open circle, HS-72Sma6A;
open square, HS-72Sma6B. Data are expressed as the
means ± S.D. of triplicate cultures. The experiment was performed
three times and similar results were obtained from each
experiment.
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To investigate whether Smad7 controls the growth arrest induced by
hBMP-2, Smad7-overexpressing clones (HS-72SM1 and HS-72SM2) and the
control clone (HS-72C4) were stimulated with hBMP-2. A control clone
(HS-72C4) showed growth arrest in response to hBMP-2. Smad7-overexpressing clones (HS-72SM1 and HS-72SM2) showed no growth
arrest in response to hBMP-2 in MTT assay (Fig. 2B). Next, we investigated whether Smad6 controls the growth arrest induced by
hBMP-2 or activin A. Smad6-expressing clones (HS-72Sma6A and HS-72Sma6B) showed no growth arrest in response to hBMP in MTT assay
(Fig. 2C). In contrast, Smad6-expressing clones (HS-72Sma6A and HS-72Sma6B) showed remarkable growth arrest in response to activin
A (Fig. 2D).
Cultivation with hBMP-2 for 24 h increased the population of cells
in the G1 phase from 16.8 to 89.9% with a reduction of those in the S phase from 76.1 to 3.3% in HS-72C4 clone. On the contrary, hBMP-2 showed no effect on the cell cycle distribution of
Smad7-overexpressing clones (HS-72SM1 and HS-72SM2) (Table I). As shown in Table
II, cultivation with activin A for
24 h increased the population of cells in the G1 phase
from 17.0 to 67.8% with a reduction of those in the S phase from 74.5 to 23.2% in the control clone (HS-72C4), indicating the cell cycle
arrest in the G1 phase. Activin A treatment also induced
the cell cycle arrest in the G1 phase in
Smad6-overexpressing clones (HS-72Sma6A and HS-72Sma6B). However,
hBMP-2 showed no effect on the cell cycle distribution of
Smad6-overexpressing clones (HS-72Sma6A and HS-72Sma6B). These results
indicate that the overexpression of Smad6 and Smad7 in HS-72 cells
abolish the cell cycle arrest in the G1 phase induced by
hBMP-2 and that overexpression of Smad6 in HS-72 cells does not abolish
the cell cycle arrest in the G1 phase induced by activin
A.
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Table I
Effects of Smad7 on hBMP-2-induced cell cycle arrest in the G1
phase
Data are expressed as the means ± S.D. of triplicate cultures.
The cells were cultured with or without hBMP-2 (50 ng/ml) for 24 h.
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Table II
Effects of Smad6 on hBMP-2- or activin A-induced cell cycle arrest in
the G1 phase
Data are expressed as the means ± S.D. of triplicate cultures. The
cells were cultured with or without hBMP-2 or activin A (50 ng/ml) for
24 h.
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Effects of Inhibitory Smads on BMP-2- and Activin A-induced Changes
in p21CIP1/WAF1--
To obtain more insight into the
mechanism by which Smad7 suppresses hBMP-2-induced cell cycle arrest in
the G1 phase, we examined the effect of Smad7 on the
BMP-2-induced expression of p21CIP1/WAF1 by immunoblot
analysis. The untreated control clone (HS-72C4) showed undetectable
levels of p21CIP1/WAF1 by immunoblot analysis (Fig.
3A). Upon exposure to hBMP-2
(50 ng/ml), expression of p21CIP1/WAF1 (21 kDa) was detected as
early as 6 h, and its level increased time dependently in the
control clone (HS-72C4). However, Smad7-overexpressing clones (HS-72SM1
and HS-72SM2) contained undetectable levels of p21CIP1/WAF1
when cultured with hBMP-2 (50 ng/ml). Furthermore, we examined the
effect of Smad7 on the hBMP-2-induced hypophosphorylation of Rb by
immunoblot analysis. As shown in Fig. 3A, hBMP-2 (50 ng/ml)
decreased the levels of hyperphosphorylated Rb in the control clone
(HS-72C4) at 12 and 24 h, resulting in a
time-dependent increase in the levels of hypophosphorylated
Rb (pRb). However, hBMP-2 showed no effect on the phosphorylation
status of Rb in Smad7-expressing clones (HS-72SM1 and HS-72SM2) during
a 24-h culture.

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Fig. 3.
Effects of ectopic expression of Smad6 and
Smad7 on the p21CIP1/WAF1 expression and Rb
hypophosphorylation in HS-72 cells. The cells were cultured with
hBMP-2 (50 ng/ml) (A and B) or activin A (50 ng/ml) (C) for the times indicated. p21CIP1/WAF1 and
Rb expression was analyzed by immunoblotting.
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Next, we examined the effect of Smad6 on hBMP-2 and the activin
A-induced expression of p21CIP1/WAF1 by immunoblot analysis.
Smad6-overexpressing clones (HS-72Sma6A and HS-72Sma6B) contained
undetectable levels of p21CIP1/WAF1 when cultured with hBMP-2
(50 ng/ml) (Fig. 3B). hBMP-2 did not change the
phosphorylation status of Rb in Smad6-overexpressing clones (HS-72Sma6A
and HS-72Sma6B) during a 24-h culture (Fig. 3B). In
contrast, an expression of p21CIP1/WAF1 was detected in 12- and
24-h cultures of the control clone (HS-72C4) and Smad6-overexpressing
clones (HS-72Sma6A and HS-72Sma6B) upon exposure to activin A (50 ng/ml) (Fig. 3C). Furthermore, activin A (50 ng/ml)
decreased the levels of hyperphosphorylated Rb in the control clone
(HS-72C4) and Smad6-overexpressing clones (HS-72Sma6A and HS-72Sma6B),
resulting in a time-dependent increase of pRb (Fig.
3C). The phosphorylation status of pRb seems to be different from the control HS-72 cells and the Smad6- and
Smad7-overexpressing cells (Fig. 3). The control HS-72C4 clone was
almost hyperphosphorylated. In contrast, the Smad7-overexpressing cells
were equally divided between hyperphosphorylated Rb and pRb, and
the Smad6-overexpressing cells were biased toward pRb. We have no ready
explanation for this phenomenon but think it is caused by the
differences in the basal growth rate of the control cells and
overexpressing cells.
Effects of Inhibitory Smads on BMP-2- and Activin A-induced
Apoptosis--
Gel electrophoresis of cellular DNA showed that
fragmented DNA was detected much less in hBMP-treated
Smad7-overexpressing clones (HS-72SM1 and HS-72SM2) and hBMP-2-treated
Smad6-overexpressing clones (HS-72Sma6A and HS-72Sma6B) than in the
control clone (HS-72C4) (Fig. 4,
A and B). In contrast, nearly the same amount of
fragmented DNA was detected in activin A-treated Smad6-overexpressing
clones (HS-72Sma6A and HS-72Sma6B) when compared with the activin
A-treated control clone (HS-72C4) (Fig. 4C). These results
indicate that an overexpression of Smad6 suppresses hBMP-2-induced
apoptosis but not activin A-induced apoptosis.

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Fig. 4.
Effects of ectopic expression of Smad6 and
Smad7 on the apoptosis in HS-72 cells. The cells were cultured
with or without hBMP-2 (50 ng/ml) (A and B) and
were cultured with or without activin A (50 ng/ml) (C) for
36 h. The DNAs were analyzed for fragmentation in an agarose gel.
Lane M, super ladder low double-stranded DNA marker kit (Gen
Sura Laboratories, Inc., Del Mar, CA).
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Effects of Inhibitory Smads on Activin A-induced Smad2
Phosphorylation--
To investigate the mechanisms by which inhibitory
Smads block activin A signals for the cell cycle arrest in the
G1 phase and apoptosis, we examined the effects of Smad6
and Smad7 on the phosphorylation status of Smad2 by activin A
stimulation with immunoblot analysis. As shown in Fig.
5A, phosphorylated Smad2 (approximately 56 kDa) was detected as early as 30 min after activin A
stimulation in the control clone (HS-72C4). However, no phosphorylated Smad2 was detected after hBMP-2 stimulation in the control clone (HS-72C4). Smad7-overexpressing clones (HS-72SM1 and HS-72SM2) contained undetectable levels of phosphorylated Smad2 when cultured with activin A (50 ng/ml) (Fig. 5B). In contrast,
phosphorylated Smad2 was detected as early as 30 min after activin A
stimulation in the control clone (HS-72C4) and Smad6-overexpressing
clones (HS-72Sma6A and HS-72Sma6B) (Fig. 5C).

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Fig. 5.
Effects of ectopic expression of Smad6 and
Smad7 on activin A-induced phosphorylation of Smad2 in HS-72
cells. A, the cells were cultured with activin A (50 ng/ml) and hBMP-2 (50 ng/ml) for the times indicated. B and
C, the cells were cultured with activin A (50 ng/ml) for the
times indicated. The phosphorylation status of Smad2 was analyzed by
immunoblotting. The antibody against phosphorylated Smad2 was raised
after immunizing rabbits with the synthetic peptide (KKKSSMS)
phosphorylated at the last two serine residues. The amount of Smad2 in
each lane was analyzed by immunoblotting with anti-mouse Smad2
monoclonal antibody (Transduction Laboratories, Lexington, KY) and is
shown in the box.
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Effects of Inhibitory Smads on BMP-2-induced Smad1/Smad5
Phosphorylation--
Next, we examined the effects of Smad6 and Smad7
expression on hBMP-2-induced phosphorylation of Smad1/Smad5 by
immunoprecipitation-immunoblot analysis using antiserum specific to
Smad1/Smad5 phosphorylated at the C-terminal SSVS motif. Untreated
control clone (HS-72C4) contained mostly unphosphorylated Smad1/Smad5.
Upon exposure to hBMP-2, the bands representing phosphorylated forms of
Smad1/Smad5 were detected as early as 0.5 h and persisted for
2 h in HS-72C4 clone. In contrast, phosphorylation of Smad1/Smad5
did not occur in either Smad6-overexpressing clone (HS-72Sma6A) or in
Smad7-overexpressing clone (HS-72SM1) for 2 h after hBMP-2
treatment (Fig. 6). These results suggest
that both Smad6 and Smad7 inhibit hBMP-2 signals by blocking the
C-terminal phosphorylation of Smad1/Smad5.

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Fig. 6.
Smad6 and Smad7 inhibit hBMP-2-mediated
Smad1/Smad5 phosphorylation in HS-72 cells. The cells were
cultured with hBMP-2 (50 ng/ml) for the times indicated and then
analyzed for the phosphorylated status of the C-terminal SSVS motif of
Smad1/Smad5 by immunoprecipitation-immunoblot analysis. The goat
anti-Smad1/Smad5 serum (N-18, Santa Cruz Biotechnology Inc.) was used
for immunoprecipitation. Anti-phosphorylated Smad1/Smad5 antibody was
raised against KKKNPISSVS phosphorylated at the last two serine
residues and used for immunoblot analysis. IgH indicates
goat immunoglobulin heavy chains.
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DISCUSSION |
We previously demonstrated that activin A induced growth arrest
and apoptosis in HS-72 cells and that the growth arrest was mediated by
induction of p21CIP1/WAF1 expression, resulting in the
inhibition of Rb phosphorylation by blocking the activity of
cyclin-dependent kinase-4 (25). In addition, we reported
that activin A showed a suppressive effect on the proliferation of
human B cell leukemias (39). In this study, we found that not only
activin A but also BMP-2 induced profound growth arrest of HS-72 cells
and the human myeloma cells line U266B1 (data not shown). These results
suggest the possible roles of activin and BMP in the regulation of
growth of B lineage cells.
Recently, we demonstrated that mRNA expression of Smad7 was induced
by activin A and that overexpression of mouse Smad7 was found to
suppress activin A-induced cell cycle arrest in the G1 phase by abolishing the activin A-induced expression of
p21CIP1/WAF1 and hypophosphorylation of Rb. Furthermore, Smad7
suppressed activin A-induced apoptosis in HS-72 cells (32). In this
study, the mRNA expression of Smad7 was induced by hBMP-2, and an
overexpression of mouse Smad7 suppressed hBMP-2-induced cell cycle
arrest in the G1 phase by abolishing the hBMP-2-induced
expression of p21CIP1/WAF1 and hypophosphorylation of Rb.
Furthermore, Smad7 suppressed hBMP-2-induced apoptosis in HS-72 cells.
These results indicate that Smad7 is an activin A- and hBMP-2-inducible
inhibitor of growth arrest and apoptosis in HS-72 cells stimulated with
activin A and hBMP-2, respectively, suggesting that Smad7 may
participate in negative feedback loops to control the growth arrest and
apoptosis induced by both activin A and hBMP-2.
It was previously reported that pathway-restricted Smads, Smad2 and
Smad3, act in the TGF-
and activin pathways (6-9). Smad1, Smad5,
and Smad8 act downstream of BMPs (10-16). In this report, we
demonstrated that Smad7 suppressed an activin A-induced phosphorylation of Smad2 and a BMP-2-induced phosphorylation of Smad1/Smad5, which occurs on two serine residues in a Ser-Ser-X-Ser motif at
the end of the C-domain of the Smads (16-18). These results suggest that Smad7 suppresses the activin A-induced growth arrest and apoptosis
by attenuating the activin A-induced signals for phosphorylation of
Smad2, and the BMP-induced growth arrest and apoptosis by attenuating the BMP-2-induced signals for phosphorylation of Smad1/Smad5.
In Xenopus embryos, ectopically expressed murine Smad7 can
antagonize activin-like signaling (28). Smad7 has also been shown to
bind to and inhibit signaling downstream of BMP receptors (29). Xenopus Smad7 is most closely related to Smad7 (74% amino
acids identical) and acts downstream of BMP-4 to modulate its activity during vertebrate embryonic patterning (40). Xenopus Smad7
was also reported to inhibit activin and BMP signalings in animal explants (41). In addition, Xenopus Smad7 was reported to
block signaling mediated by the activin receptor ALK-4 and inhibit
BMP-4 signaling (42). Taken together, these findings indicate that Smad7 may act as a general inhibitor of the TGF-
family and support our results showing that Smad7 inhibited the growth arrest and apoptosis induced by both activin A and hBMP-2.
Afrakhte et al. (43) reported that the mRNA expression
of Smad6 is induced by multiple stimuli derived from the various TGF-
family members. In this study, we also found that the mRNA expression of Smad6 was induced by both activin A and hBMP-2 in HS-72
cells. Moreover, we demonstrated that Smad6 was an hBMP-2-inducible inhibitor of the hBMP-2-induced growth arrest and apoptosis in HS-72
cells, suggesting that Smad6 may participate in a negative feedback
loop to control the growth arrest and apoptosis induced by hBMP-2 in B
lineage cells.
On the contrary, an overexpression of Smad6 was not found to suppress
the growth arrest and apoptosis induced by activin A. In addition, we
demonstrated that Smad6 blocked a BMP-2-induced phosphorylation of
Smad1/Smad5, suggesting that Smad6 suppresses a BMP-2-induced growth
arrest and apoptosis by attenuating the BMP-2-induced signals for
phosphorylation of Smad1/Smad5. However, Smad6 did not suppress activin
A-induced growth arrest and apoptosis because of its incapability for
attenuating activin A-induced signals for phosphorylation of Smad2.
These results suggest that Smad6 is a ligand-specific inhibitor of
growth arrest and apoptosis in HS-72 cells. Thus, Smad6 and Smad7
exhibit differential inhibitory activities toward BMP- and
activin-induced responses in mouse B cells.
We examined the endogenous protein levels of Smad6 and Smad7 in HS-72
cells by immunoblotting. Both Smad6 and Smad7 proteins were detected in
Smad6- and Smad7-overexpressing cells, respectively. However, the
endogenous Smad6 and Smad7 proteins were not detected, even when HS-72
cells were stimulated with activin A and BMP-2 for 12 and 24 h
(data not shown). Whether Smad6 and Smad7 have a role in negative
feedback control in myeloma cells will be the subject of future studies.