Differential Inhibition of Smad6 and Smad7 on Bone Morphogenetic Protein- and Activin-mediated Growth Arrest and Apoptosis in B Cells*

Akira IshisakiDagger , Kenji Yamato§, Shinichi HashimotoDagger , Atsuhito Nakaoparallel , Kiyoshi Tamaki**, Koji NonakaDagger , Peter ten Dijke**, Hiromu SuginoDagger Dagger , and Tatsuji NishiharaDagger §§

From the Dagger  Department of Oral Science, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan, the § Department of Molecular Cellular Oncology/Microbiology and the  First Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan, the parallel  Department of Medicine, Chiba University, School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba City, Chiba 260-0856, Japan, the Dagger Dagger  Institute for Enzyme Research, University of Tokushima, 3-18-15 Kuramoto, Tokushima 770, Japan, and the ** Ludwig Institute for Cancer Research, Box 595, Biomedical Center, S-751 24 Uppsala, Sweden

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

Smad6 and Smad7 prevent ligand-induced activation of signal-transducing Smad proteins in the transforming growth factor-beta family. Here we demonstrate that both Smad6 and Smad7 are human bone morphogenetic protein-2 (hBMP-2)-inducible antagonists of hBMP-2-induced growth arrest and apoptosis in mouse B cell hybridoma HS-72 cells. Moreover, we confirmed that the ectopic expressions of Smad6 and Smad7 inhibited the hBMP-2-induced Smad1/Smad5 phosphorylation. We previously reported that Smad7 is an activin A-inducible antagonist of activin A-induced growth arrest and apoptosis in HS-72 cells. Interestingly, although mRNA expression of Smad6 was induced by activin A in HS-72 cells, Smad6 showed no antagonistic effect on activin A-induced growth arrest and apoptosis. Moreover, we found that the ectopic expression of Smad7, but not Smad6, inhibited the activin A-induced Smad2 phosphorylation in HS-72 cells. Thus, Smad6 and Smad7 exhibit differential inhibitory effects in bone morphogenetic protein-2- and activin A-mediated signaling in B lineage cells.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Transforming growth factor-beta (TGF-beta )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-beta 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-beta family signaling) (19-21). This hetero-oligomeric Smad complex then translocates to the nucleus and modulates transcription of TGF-beta 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-beta family signaling (26-30). Smad7 forms stable interactions with the activated TGF-beta 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-beta 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-beta signal for phosphorylation of Smad2 in COS-7 cells and suppresses the activation of p3TP-Lux by TGF-beta 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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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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 [alpha -32P]dCTP using a MultiprimeTM DNA labeling system (Amersham Pharmacia Biotech). Hybridization and washing were performed as described previously (38).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

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.

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.

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.

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).

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.

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.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-beta 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-beta 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-beta 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.

    ACKNOWLEDGEMENTS

We thank Dr. K. Miyazono for kindly providing us with plasmid pcDNA3-FLAG-Smad6, Dr. Y. Eto (Ajinomoto Co., Ltd.) for activin A, and Yamanouchi Pharmaceutical Co., Ltd. for hBMP-2.

    FOOTNOTES

* This work was supported in part by a grant from the Japan Health Science Foundation and by grants-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§§ To whom correspondence should be addressed: Dept. of Oral Science, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan. Tel.: 81-3-5285-1111 (ext. 2220); Fax: 81-3-5285-1172; E-mail: tatsujin{at}nih.go.jp.

    ABBREVIATIONS

The abbreviations used are: TGF-beta , transforming growth factor-beta ; BMP, bone morphogenetic protein; hBMP-2, human bone morphogenetic protein-2; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Rb, retinoblastoma protein; pRb, hypophosphorylated retinoblastoma protein.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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