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Oncogene (1998) 17, 1743 ± 1747 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc SHORT REPORT Induction of apoptosis by Smad3 and down-regulation of Smad3 expression in response to TGF-b in human normal lung epithelial cells Kiyoshi Yanagisawa1,5, Hirotaka Osada1,2, Akira Masuda1, Masashi Kondo1,5, Toshiko Saito1, Yasushi Yatabe4, Kenzo Takagi5, Toshitada Takahashi3 and Takashi Takahashi1,2 1 Laboratory of Ultrastructure Research, 2Pathophysiology Unit and 3Laboratory of Immunology, Aichi Cancer Center Research Institute, Kanokoden, Chikusa-ku, Nagoya 464; 4Department of Pathology and Clinical Laboratories, Aichi Cancer Center Hospital, Kanokoden, Chikusa-ku, Nagoya 464; 5Internal Medicine 2, Nagoya University School of Medicine, Tsurumai-cho, Showa-ku, Nagoya 466, Japan Smad family members are essential intracellular signaling components of the transforming growth factor-beta (TGF-b) superfamily involved in a range of biological activities. Two highly homologous molecules, Smad2 and Smad3, have so far been identi®ed as receptor-activated Smads for TGF-b signaling and have become the focus of intensive studies. However, no de®nite dierences in regulation or function have been established between these TGF-b signaling molecules. In the present study, we show that the expression of Smad3, but not its close relative, Smad2, is down-regulated by TGF-b mediated signals themselves in human lung epithelial cells. This down-regulation of Smad3 by TGF-b treatment did not appear to result from shortening of the half-life of Smad3 mRNA. Constitutive expression of Smad3 in the presence of TGF-b induced apoptotic cell death, with an adverse eect on the cell growth of human lung epithelial cells. Apoptotic cell death could also be induced by forced expression of Smad2 in the presence of TGF-b, but less eciently than by that of Smad3. These ®ndings clearly de®ne the distinctions between Smad2 and Smad3 for the ®rst time in that a qualitative dierence was observed with regard to the regulation of their expression in response to TGF-b, while Smad2 and Smad3 appeared to have quantitatively dierent capabilities regarding the induction of apoptotic cell death in human lung epithelial cells. Keywords: Smad; TGF-b; apoptosis; down-regulation; lung The TGF-b superfamily members, including activin, bone morphogenetic protein and TGF-b, play important regulatory roles in cell growth, morphogenesis, cell dierentiation, and apoptosis (Kingsley, 1994; Massague, 1996; Roberts and Sporn, 1993). Signaling mediators downstream of the receptors for the TGFb superfamily were recently identi®ed in a variety of organisms, and termed `Smad' in vertebrates (Derynck et al., 1996). While seven Smad genes have been reported in the literature thus far, they can be classi®ed into three types according to their functions, i.e., receptor-activated Smads (Smad1 and Smad5 for BMP; Smad2 and Smad3 for TGF-b and activin), co-Smad Correspondence: Takashi Takahashi Received 28 January 1998; revised 14 April 1998; accepted 15 April 1998 (Smad4) and anti-Smads (Smad6 and Smad7) (Baker et al., 1996; Derynck et al., 1996; Eppert et al., 1996; Gra et al., 1996; Hahn et al., 1996; Hayashi et al., 1997; Hoodless et al., 1996; Lechleider et al., 1996; Liu et al., 1996; Riggins et al., 1996; Thomsen, 1996; Topper et al., 1997; Yingling et al., 1996; Zhang et al., 1996). Smad2 and Smad3, the receptor-activated, TGFb signaling Smads, are known to be highly homologous with regard to amino acid sequence and structural characteristics, and both have been found to mediate TGF-b and activin signals by forming heteromers with Smad4 (Eppert et al., 1996; Gra et al., 1996; Riggins et al., 1996; Wrana and Pawson, 1997; Zhang et al., 1996). Despite extensive studies of the Smad gene family, however, no de®nitive dierences in the regulations or functions of these receptor-activated Smads have been identi®ed. The loss of sensitivity to TGF-b has been shown to be common in human cancers (Roberts and Sporn, 1993). While Smad4 was ®rst isolated as a pancreatic tumor suppressor gene (Hahn et al., 1996), mutations in the Smad2 and Smad4 genes have also been reported in other common cancers of adults (Eppert et al., 1996; Nagatake et al., 1996; Riggins et al., 1996; Uchida et al., 1996). Our ®ndings that Smad2 and Smad4 both at 18q21 were somatically mutated in a fraction of human lung cancers (Nagatake et al., 1996; Uchida et al., 1996) suggested that members of the Smad gene family may play a role in the pathogenesis of lung cancer, since normal lung epithelial cells are known to exhibit a growth-inhibitory response to TGF-b (Masuda et al., 1997; Masui et al., 1986). The present study was undertaken to gain further insights into the regulation and roles of the TGF-b signaling pathway in lung epithelial cells. Using BEAS2B (Reddel et al., 1988) and HPL1A lung epithelial cell lines (Masuda et al., 1997), we ®rst examined whether various external stimuli can alter the expression levels of TGF-b signaling Smads, i.e., Smad2, Smad3 and Smad4. Northern blot analysis showed readily detectable expression of all three Smads in both BEAS2B and HPL1A (Figure 1a). Addition of 1 ng/ml of TGF-b to the culture media resulted in a signi®cant reduction of Smad3 mRNA expression in both cell lines, whereas expression levels of Smad2 and Smad4 remained virtually unaltered. In contrast, treatment with 10 ng/ml of epidermal growth factor (EGF) or use of various concentrations (0.1, 1, 10%) of fetal calf serum had no eect on either the expression of Smad3 or that of Smad2 and Smad4 TGF-û signaling Smads in lung epithelial cells K Yanagisawa et al 1744 signal down-regulates Smad3 mRNA expression, we examined the half-lives of Smad3 mRNA in the presence or absence of TGF-b in BEAS2B. BEAS2B cells were cultured for 8 h in the presence or absence of 1 ng/ml of TGF-b and then chased in the presence of 5 mg/ml of actinomycin D together with 1 ng/ml of TGF-b for up to 12 h. Northern blot analysis using RNAs harvested at various time points revealed that the half-life of Smad3 mRNA was 4.7 h with, and 5.1 h without TGF-b treatment, indicating that TGFb treatment did not signi®cantly alter the stability of Smad3 mRNA (Figure 2). These results suggested that the reduction of Smad3 mRNA levels may be mainly caused by the decrease in the Smad3 gene transcription. Smad2 mRNA was found to have a considerably longer half-life (12.5 h), and did not exhibit any change in its half-life after the addition of TGF-b. The next question was what the biological outcome would be of constitutive expression of Smad3 in BEAS2B cells, in which TGF-b was found to rapidly down-regulate Smad3 expression. A tetracycline-o gene expression system, in which addition of tetracycline to the culture media arrests gene transcription (Ookawa et al., 1997), was employed for this purpose. BEAS2B-tet-Smad3 stable transfecFigure 1 Northern blot analysis of Smad2, Smad3 and Smad4 in response to external stimuli in two immortalized human normal lung epithelial cell lines, BEAS2B and HPL1A. (a) Marked reduction of Smad3 mRNA is observed only in the presence of TGF-b, whereas no noticeable changes are detected as a result of the addition of EGF or of either low (0.1%) or high (10%) serum concentrations (data not shown). In contrast Smad2 and Smad4 expressions remain virtually unchanged under all experimental conditions. (b) Reduction of Smad3 is detectable as early as 4 h after addition of TGF-b. (a) and (b) BEAS2B and HPL1A cells were maintained in Ham's F12 medium buered with 15 mM HEPES (pH 7.3) and supplemented with bovine insulin (5 mg/ml), human transferrin (5 mg/ml), 1077 M hydrocortisone, 2610710 M triiode thyronine, penicillin (100 IU/ml), streptomycin (100 mg/ml) and 1% FCS. To examine the possible eects of external stimuli on Smad2, Smad3 and Smad4 mRNA expression, BEAS2B and HPL1A cells were cultured for 6 days in the presence of 1 ng/ml TGF-b (Wako, Osaka, Japan) or 10 ng/ml EGF (Austral Biologicals, San Ramon, CA). In addition, the eects of lower and higher serum concentrations (0.1 and 10% FCS) were also examined. Extraction of total cellular RNA and Northern blot analysis was performed with the aid of probes generated by the following oligonucleotide primers. Smad2S (sense), 5'-AGCTCTAGATGGCTTGCTGCCTTTGGTAAG; Smad2AS (antisense), 5'-AGCGAATTCAAGTCTTTTCCATGGGACTTG; Smad3S (sense), 5'-AGCTCTAGAACTCAAGAAGACGGGGCAGCT; Smad3AS (antisense), 5'-AGCGAATTCCTACTTGGTGTCGTACCTGCG; Smad4S (sense), 5'-AGCAAGCTTGCTTCAGAAATTGGAGAC; Smad4AS (antisense), 5'-AGCGGATCCCCATCCTGATAAGGTTAAGGGC. Ethidium bromide staining of the gels is shown as a control for RNA loading (Figure 1a and data not shown for serum). Northern blot analysis using RNAs harvested at various time points revealed that down-regulation of Smad3 expression can be observed as early as 4 h after addition of 1 ng/ml of TGF-b, with a 50% reduction at 9.3 h (Figure 1b). These ®ndings clearly indicated for the ®rst time that expression of a TGF-b signaling molecule, Smad3, but not its close relative, Smad2, is regulated by a TGF-b mediated signal in human lung epithelial cells. As an initial step towards elucidation of the possible mechanism by which the TGF-b elicited Figure 2 Northern blot analysis for determining of the half-lives of Smad2 and Smad3 mRNA. (a) and (b) Stability of neither Smad2 nor Smad3 mRNAs is aected by the addition of TGF-b. (a) and (b) BEAS2B cells were cultured for 8 h in the presence or absence of 1 ng/ml of TGF-b and chased in the presence of 5 mg/ ml of actinomycin D (Sigma Chemical Co., St. Louis, MO) together with 1 ng/ml of TGF-b for up to 12 h. RNAs were extracted at various time points after the addition of actinomycin D and subjected to Northern blot analysis using the cDNA probes described above. The amounts of Smad2 and Smad3 mRNAs were measured with a BAS2000 image analyser (Fuji Photo Film Co., Ltd., Toyko, Japan). Ethidium bromide staining of the gels is shown as a control for RNA loading TGF-û signaling Smads in lung epithelial cells K Yanagisawa et al tants (clones 3 and 10) were generated by transfection of pTA-hyg, which contained a fusion gene (tTA) between the tetracycline responsive repressor and the activation domain of herpes simplex virus VP16 protein, followed by transfection of pT2GN-Smad3 harboring wild-type Smad3 cDNA in a tTA responsive pT2GN (Zhang et al., 1996). As shown in Figure 3a, the induction of Smad3 in BEAS2B-tet-Smad3 clone 3 by removal of tetracycline resulted in a moderately inhibitory eect on cell growth in the absence of TGF-b (tet7, TGF-b7), while TGF-b treatment alone also caused modest growth inhibition (tet+, Figure 3 The eect of constitutive expression of Smad3 in the presence of TGF-b on cell proliferation and viability. (a) Constitutive Smad3 expression in the presence of TGF-b results in a decrease in the number of cells. (b) Apoptotic cell deaths are observed only under the Smad3-induced and TGF-b-added conditions. (a) A tetracycline-o gene expression system, in which the addition of tetracycline (Wako, Osaka, Japan) to the culture media arrests gene transcription, was used to regulate expression of Smad3. BEAS2B-tet-Smad3 stable transfectants (clones 3 and 10) were generated by sequential transfection of 1.25 mg of pTA-hyg and then of 2 mg of pT2GN-Smad3 using the DMRIE-C method (Life Technologies, Inc., Gaithersburg, MD). The transfected cells were selected using 100 mg/ml hygromycin B (Sigma Chemical Co.) and 200 mg/ml G418, respectively. BEAS2B-tet-Smad3 cells were seeded in 96-well cell culture plates at 5000 cells/well in 100 ml of medium containing 1 mg/ml of tetracycline and preincubated at 378C for 24 h. Smad3 was then induced by removal of tetracycline and cells were exposed to 1 ng/ml of TGF-b. MTS assay was performed at various time points using the TetraColor One cell proliferation assay system (Seikagaku Corp., Tokyo, Japan). Each data point represents the mean+s.d. of triplicate samples. Three independent experiments gave similar results. (b) Apoptotic cell deaths were visualized by the TUNEL method using the in situ cell death detection kit (Boehringer Mannheim Gmbh, Mannheim, Germany) TGF-b+). Notably distinct results were obtained when cells were placed under constitutive Smad3 expression in the presence of TGF-b (tet7, TGFb+). In contrast to the gradual increase in the number of cells with tet+, TGF-b+ or tet7, TGFb7 treatment, the number of cells decreased steadily under tet7, TGF-b+ condition and did not recover, showing the signi®cantly adverse eect of constitutive Smad3 expression in the presence of TGF-b. The terminal deoxynucleotidyl transferase-mediated dUTPbiotin nick end labeling (TUNEL) method was employed to examine whether apoptotic cell death plays a role in this process. TUNEL staining-positive cells were frequently observed only when Smad3 was constitutively expressed in the presence of TGF-b (Figure 3b). Similar results were also obtained for BEAS2B-tet-Smad3 clone 10 (data not shown). Constitutive Smad3 expression in the presence of TGF-b thus appeared to adversely aect the growth of lung epithelial cells by inducing apoptotic cell death. We next wanted to know whether there is a dierence in the eect on cell proliferation and viability between Smad2 and Smad3 proteins. For this purpose, a tetracycline-o gene expression system was employed to regulate the expression of either Smad2 or Smad3, while a luciferase assay system was used as an indicator of viable cells because good correlation was observed between number of cells and luciferase activity (correlation coecient=0.974). BEAS2B-tet cells were transfected with 2 mg of pT2GN-Smad2 or pT2GN-Smad3 together with 0.2 mg of pCMV-luc carrying the cytomegalovirus (CMV) promoter-driven luciferase gene. Cells were subsequently refed with fresh media containing tetracycline and TGF-b. Luciferase activity was Figure 4 The eect on cell proliferation and viability of the constitutive expression of Smad2 and Smad3 in the presence or absence of TGF-b. Constitutive Smad3 expression in the presence of TGF-b results in a signi®cant decrease in luciferase activity during the incubation period, indicating a continued decrease in the number of cells. Note that luciferase activities recover gradually under other conditions. A tetracycline-o gene expression system was employed to regulate the expression of either Smad2 or Smad3, while luciferase activity was measured as an indicator of viability of transfected cells by using the Luciferase Assay System (Promega, Madison, WI). Values represent luciferase activity relative to that of each of the control transfectants incubated under tetracycline (+) and TGF-b (7) condition and are shown as mean+s.d. of triplicate dishes. Similar results were obtained in three independent experiments 1745 TGF-û signaling Smads in lung epithelial cells K Yanagisawa et al 1746 measured 24 and 48 h later. As shown in Figure 4, induction of Smad2 and Smad3 by removal of tetracycline in the absence of TGF-b (tet7, TGFb7) had a moderately inhibitory eect on cell growth as indicated by the decrease in luciferase activity. TGF-b treatment alone without Smad2 or Smad3 induction (tet+, TGF-b+) also showed a moderately inhibitory eect on cell growth. However, in these conditions luciferase activity increased gradually, indicating that the number of cells were still growing. It is noteworthy that when Smad3 was constitutively expressed in the presence of TGF-b (tet7, TGF-b+), luciferase activity decreased significantly from 24 to 48 h, indicating a continued decrease in the number of cells. In contrast, the induction of Smad2 in the presence of TGF-b inhibited cell growth, but still allowed for a gradual increase in the number of cells, indicating a much less pronounced adverse eect than that of Smad3. We further examined whether there might be any dierences between Smad2 and Smad3 in their capacity to induce apoptosis. BEAS2B cells were cotransfected with 2 mg of either pCMV-FlagSmad2 or pcDNA3-FlagSmad3 driven by the constitutive CMV promoter and tagged with Flag, together with 0.2 mg of pCMV-CD20. Similar levels of Smad2 and Smad3 expression were con®rmed by Western blot analysis using the anti-FlagM2 antibody (IBI, Eastman Kodak, Rochester, NY). Apoptotic cells among the CD20-positive cells were detected by using the TUNEL method. Transfected cells were cultured in the presence or absence of TGF-b for 48 h. As expected, Smad3 transfectants maintained in the presence of TGF-b showed a marked induction of apoptotic cell death, and 55% of the CD20-positive cells were found to be TUNEL-staining positive (Figure 5). Smad2 transfectants cultured in the presence of TGF-b were also rendered to be apoptotic but considerably less signi®cantly. These results showed that both Smad2 and Smad3 were capable of inducing apoptotic cell death of lung epithelial cells, at least when constitutively overexpressed in the presence of TGF-b, while Smad3 could induce this biological response more eciently than Smad2. In the present study, we demonstrated that expression of a TGF-b signaling mediator, Smad3, but not its close relative, Smad2, was down-regulated by the TGF-b mediated signals themselves. Reduction of Smad3 mRNA levels did not appear to result from shortening of its half-life. We could also show that forced constitutive expression of Smad3 in the presence of TGF-b induced marked apoptotic cell death in human normal lung epithelial cells. One could speculate that down-regulation of Smad3 expression in response to TGF-b might be a negative feedback mechanism by which lung cells can avoid excess TGF-b signals, although the underlying assumption that protein expression of Smad3 is indeed diminished in response to TGF-b needs to be con®rmed. The present study thus shows clear distinctions between Smad2 and Smad3, in that a qualitative dierence was observed with regard to the regulation of their mRNA expression in response to TGF-b, while Smad2 and Smad3 appeared to be quantitatively dierent in their adverse eect on cell growth of lung epithelial cells. We note that Zhang et al. (1996) and Yingling et al. (1997) previously showed that co-overexpression of Smad3/Smad4, but not Smad2/Smad4, induced transcriptional activation of plasminogen activator inhibitor-1 promoter, while Imamura et al. (1997) reported selective binding of Smad6 to Smad2. Taken together, these ®ndings suggest that these two highly related Smads may have distinct functional roles. Since TGF-b-mediated signals are known to play important roles in lung development, future studies are warranted to investigate distinctions between in situ expression of Smad2 and Smad3 during the developmental process. In addition, although our extensive search did not reveal any Smad3 mutations in lung cancers (unpublished observation), in contrast to the occurrence of Smad2 mutations (Uchida et al., 1996), it would also be interesting to examine whether there are any alterations in the regulation of Smad3 expression in lung cancer cells. Figure 5 Apoptotic cell death is induced by the forced expression of both Smad2 and Smad3 in the presence of TGFb, while Smad3 appears more eective for induction of this biological response. BEAS2B cells were co-transfected with 2 mg of pCMV-FlagSmad2 and pcDNA3-FlagSmad3 driven by the constitutive CMV promoter and tagged with Flag, together with 0.2 mg of pCMV-CD20. The construct of Smad2 was generated by the following oligonucleotide primers: Smad2 (sense), 5'AGCTCTAGATGGCTTGCTGCCTTTGGTAAG; Smad2AS (antisense), 5'-AGCGAATTCAAGTCTTTTCCATGGGACTTG. Five hours after transfection, cells were placed in the conditions indicated with the use of 1 mg/ml of tetracycline and 1 ng/ml of TGF-b. Forty-eight hours later, transfected cells were examined by Western blot analysis to con®rm equal expression of Smad2 and Smad3 using 50 mg of the solubilized proteins and antiFlagM2 antibody (Eastman, Kodak). Transfected cells were also examined by the TUNEL method. In brief, cells were ®xed in PBS containing 4% paraformaldehyde for 30 min at room temperature and detected by using the anti-CD20 antibody (DAKO A/S, Glostrup, Denmark) and subsequent application of biotinylated anti-mouse IgG (Vector, Burlingame, CA) and tetramethylrodamine-isothiocyanate-isomer R conjugated-streptavidin (Cosmo Bio Co. Ltd., Tokyo, Japan). Apoptotic cell deaths were examined as described in the legend for Figure 3. The number of TUNEL-stained CD20-positive cells was determined by counting at least 1000 CD20-positive cells. Values represent apoptotic cells as a percentage of CD20-positive cells and are shown as means+s.d. of triplicate dishes. 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