Smad7 and Smad6 Differentially Modulate Transforming Growth Factor β-induced Inhibition of Embryonic Lung Morphogenesis*

Transforming growth factors β (TGF-β) are known negative regulators of lung development, and excessive TGF-β production has been noted in pulmonary hypoplasia associated with lung fibrosis. Inhibitory Smad7 was recently identified to antagonize TGF-β family signaling by interfering with the activation of TGF-β signal-transducing Smad complexes. To investigate whether Smad7 can regulate TGF-β-induced inhibition of lung morphogenesis, ectopic overexpression of Smad7 was introduced into embryonic mouse lungs in culture using a recombinant adenovirus containing Smad7 cDNA. Although exogenous TGF-β efficiently reduced epithelial lung branching morphogenesis in control virus-infected lung culture, TGF-β-induced branching inhibition was abolished after epithelial transfer of the Smad7 gene into lungs in culture. Smad7 also prevented TGF-β-mediated down-regulation of surfactant protein C gene expression, a marker of bronchial epithelial differentiation, in cultured embryonic lungs. Moreover, we found that Smad7 transgene expression blocked Smad2 phosphorylation induced by exogenous TGF-β ligand in lung culture, indicating that Smad7 exerts its inhibitory effect on both lung growth and epithelial cell differentiation through modulation of TGF-β pathway-restricted Smad activity. However, the above anti-TGF-β signal transduction effects were not observed in cultured embryonic lungs with Smad6 adenoviral gene transfer, suggesting that Smad7 and Smad6 differentially regulate TGF-β signaling in developing lungs. Our data therefore provide direct evidence that Smad7, but not Smad6, prevents TGF-β-mediated inhibition of both lung branching morphogenesis and cytodifferentiation, establishing the mechanistic basis for Smad7 as a novel target to ameliorate aberrant TGF-β signaling during lung development, injury, and repair.

Transforming growth factor ␤ (TGF-␤) 1 family members elicit a diverse range of cellular responses including cell proliferation, differentiation, migration, organization, and apoptosis (1). TGF-␤ signaling occurs via ligand-initiated complex formation of a heteromeric serine-threonine kinase receptor complex, following which the TGF-␤ type I receptor is phosphorylated by the constitutively active TGF-␤ type II receptor (2). The activated type I receptor then propagates the signal through transient interaction with, and phosphorylation of, TGF-␤ pathway-restricted Smad2 and Smad3 proteins (3). Smad4 forms hetero-oligomeric complexes with Smad2 or Smad3 proteins, which translocate into the cell nucleus and subsequently activate transcriptional responses (4 -6).
The TGF-␤-signaling pathway has been implicated in the normal temporo-spatial pattern of lung morphogenesis and concomitant pulmonary-specific gene expression. Both addition of exogenous TGF-␤1 and TGF-␤2 to embryonic mouse lungs in culture and organ-specific overexpression of TGF-␤1 in transgenic mice bearing a chimeric surfactant protein-C (SP-C) promoter-directed TGF-␤1 expression construct result in hypoplastic phenotypes (7)(8)(9), indicating that exogenous TGF-␤ ligands exert negative regulatory influences on lung development. On the other hand, abrogation of TGF-␤ type II receptor signaling, either with antisense oligodeoxynucleotides or with pulmonary epithelium-specific overexpression of dominantnegative TGF-␤ type II receptor, stimulates lung morphogenesis in culture and prevents TGF-␤-induced inhibition of epithelial differentiation marker genes such as SP-C (10,11), further supporting the conclusion that endogenous TGF-␤ signaling negatively regulates lung morphogenesis and cytodifferentiation.
Although TGF-␤ signaling is an important negative modulator during lung development, aberrant expression of TGF-␤ has been noted in the pathogenesis of lung diseases such as lung fibrosis (12). Prolonged overproduction of TGF-␤ is known to induce a chronic fibrotic lung injury in rats as well as in the bleomycin model of chronic fibrosis (13,14). Alveolar hypoplasia, a main sequela of neonatal hyperoxia, is also associated with high levels of TGF-␤ activity in premature lungs (15). Pulmonary fibrosis is also a prominent feature of bronchopulmonary dysplasia, one of the primary causes of neonatal mortality and morbidity, and elevated concentrations of TGF-␤1 have been found in the bronchoalveolar lavage fluid of human premature infants with bronchopulmonary dysplasia (16). Therefore, excessive TGF-␤ signaling appears to adversely disrupt the orderly temporo-spatial molecular cascades that normally instruct lung growth, differentiation, and development.
Recent studies have provided new understanding of how TGF-␤ signaling is "switched off." Both Smad6 and Smad7 have been identified to form stable associations with the activated TGF-␤ type I receptor, thereby preventing the latter from binding to and activating cognate pathway-restricted Smads (17,18). The inhibitory Smad6 and Smad7 diverge structurally from other Smad family members by the lack of the C-terminal phosphorylation sites (4). The expression of inhibitory Smads are, themselves, induced by TGF-␤ stimulation, suggesting that the inhibitory Smads attenuate TGF-␤ signal transduction through an intracellular negative feedback loop (18).
The discovery of inhibitory Smads suggests novel functional mechanisms to modulate TGF-␤-mediated signal transduction during lung growth and development. We have recently shown that Smad7 gene is predominantly expressed in distal airway epithelium and that abrogation of endogenous Smad7 gene expression using a Smad7 antisense oligodeoxynucleotide increased the TGF-␤-mediated negative effect on embryonic mouse lung branching morphogenesis in culture (19). To test further the functional role of inhibitory Smads in developing lungs, we examined the biological effect of exogenous Smad6 and Smad7 introduced by recombinant adenoviruses into embryonic mouse lungs in serumless, chemically defined culture. Our findings demonstrated that gene transfer of Smad7 abolished TGF-␤induced inhibition of both lung branching morphogenesis and cytodifferentiation, suggesting that modulation of Smad7 expression plays an important biological role during lung organogenesis. In contrast, Smad6 overexpression in lungs failed to antagonize TGF-␤-induced lung branching inhibition, indicating that Smad7 and Smad6 exert differential regulatory effects during lung morphogenesis. Smad7 thus appears to be a novel molecule to reduce excessive TGF-␤ signaling during lung morphogenesis, development, injury, and repair.

MATERIALS AND METHODS
Adenovectors and Their Administration to Mouse Lung-Replicationdeficient recombinant adenoviruses expressing full-length murine Smad7 (AdSmad7) and Smad6 (AdSmad6) under the control of a murine cytomegalovirus promoter were used to locally overexpress Smad7 and Smad6, respectively, in the embryonic mouse lungs. Both Smad7 and Smad6 adenovectors were generously provided by Dr. Makiko Fujii (Bethesda, MD), and the FLAG-epitope sequence was fused to the N terminus of Smad7 or Smad6 cDNA. AdSmad7 or AdSmad6 at a dose of at least 1 ϫ 10 10 plaque-forming units/ml was prepared and purified from human 293 cell line, as described previously (20).
Timed pregnant Swiss-Webster mice were sacrificed on postcoitum day 11 (E11), and the lung primordia were isolated from embryos by micro-dissection. AdSmad7, AdSmad6, or a control vector containing no exogenous gene in phosphate-buffered saline was intratracheally microinjected into the embryonic lung airway as we have previously reported (11). High titer adenovirus in a volume of 50 nl was used for each lung explant until the whole embryonic respiratory tract was filled with injected adenoviral solution. Injected lungs were incubated for 1 h at room temperature for viral transfection prior to culture.
Lung Organ Explant Culture-Adenovirus-infected lung explants were cultured under serum-free, chemically defined conditions as described previously (21). Known concentrations of TGF-␤ ligand (R & D Systems, Minneapolis, MN) were added exogenously to culture medium as needed. The cultures were maintained in 100% humidity with 5% CO 2 supplied for 4 days. The medium was changed every other day.
Measurement of Lung Branching Morphogenesis-Since branching morphogenesis per se is the key bioassay read-out for evaluating the functional role of Smad7 and Smad6 during early lung organogenesis, three independent measurements of branching morphogenesis were used as follows: (i) the number of airway generations from the trachea to the most distal branch of the longest visible airway; (ii) the number of air sacs visible around the periphery of the lung explants; and (iii) computerized pattern recognition analysis of the number of individual terminal respiratory units in the whole explant. These analyses were performed on whole mounts of lung explants without knowledge of the assay conditions, using transillumination to visualize structures and photomicroscopy to record permanent images. Paraffin-embedded sections of cultured lung tissue after fixation were also examined to verify the counting of terminal branching.
RNA Extraction, Reverse Transcription, and Competitive PCR Quantification-Total RNA was extracted from cultured lung explants using guanidinium thiocyanate following homogenization as we have documented earlier (10). Extracted total RNA was subsequently reversetranscribed using oligo(dT) primer and Moloney murine leukemia virus-reverse transcriptase (Life Technologies, Inc.). The resultant cDNAs were used for competitive PCR assay.
Methodology for specific pulmonary gene mRNA quantification using competitive PCR has been documented elsewhere (10). For Smad6, a set of primers (primers 1 and 2, Fig. 1A) was designed for mouse Smad6 to amplify a PCR product 303 bp in size (22). To generate competitor cDNA for Smad6 competitive PCR assay, the above primer sequences (primers 1 and 2) were engineered into a heterologous DNA fragment using the same strategy as we have previously described (10). Therefore, both the Smad6 cDNA and the Smad6 competitor share the same set of primers in the Smad6-competitive PCR amplification. The Smad6 competitor was 370 bp in length. Both Smad6 and its competitor PCR products were DNA sequenced to ensure their sequence accuracy. The same amount of Smad6 competitor was co-amplified with serial dilutions of Smad6 cDNA, and a linear relationship between initial mouse Smad6 concentration and the logarithmic ratio of Smad6 target to its competitor band intensities was obtained upon densitometric scanning of the ethidium bromide-stained gels (Fig. 1, B and C). Competitive PCR assays for other genes used in this report were developed in a similar manner as that for Smad6.
Western Blot Analysis-Cultured embryonic lungs were lysed immediately in SDS buffer containing protease inhibitors and sodium fluoride. Equal amounts of total lung protein (50 g) from each assayed sample were used for chemiluminescent Western analysis (Roche Molecular Biochemicals) on Immobilon-P membrane (Millipore, Bedford, MA) as described (10). Immunoblotting was performed using antibodies against mouse Smad2 (Transduction Laboratories, Lexington, KY), phosphorylated Smad2 (a gift from Dr. Carl-Henrik Heldin, Uppsala, Sweden), and ␤-actin (Santa Cruz Biotechnology, Santa Cruz, CA) at recommended dilutions (18). An anti-FLAG M2 monoclonal antibody (Sigma) was purchased to detect adenoviral transgene expression in cultured lungs using procedures suggested by the manufacturer.
Immunohistochemistry-Cultured mouse lungs were fixed in 4% paraformaldehyde for 3 h at room temperature and immediately embedded into paraffin. Lung tissues were prepared into 5-m thick FIG. 1. Competitive PCR assay for Smad6 mRNA quantification. A, a pair of primers (primers 1 and 2, hatched boxes) was designed to mplify both Smad6 cDNA and its competitor, and the corresponding PCR products were 303 and 370 bp in length, respectively. B, a fixed amount of Smad6 competitor was used to co-amplify with a 1:2 serial dilution of Smad6 cDNA, and the resultant electrophoretic pattern displayed a reverse correlation of intensities between Smad6 cDNA and Smad6 competitor bands. C, a linear relationship (open squares) was derived from electrophoretic pattern with linearity (R 2 Ͼ0.95) following densitometric analysis of DNA band intensities, and a similar line with identical slope value (dashed line) was obtained when different amounts of reverse-transcribed total RNA from cultured mouse lungs replaced Smad6 cDNA.
sections on HistoGrip-coated microscopic slides (Zymed Laboratories Inc., South San Francisco, CA). The expression of exogenous Smad7 or Smad6 transgene derived from the adenoviral vector was assessed by immunohistochemical staining, using a goat polyclonal anti-FLAG antibody (Santa Cruz Biotechnology) and normal goat IgG (negative control) as described previously (11).
Data Analysis-The data in the present study were expressed as mean Ϯ S.D., and the significance of variances between means was evaluated by Student's t test. p values less than 0.05 were considered to be statistically significant.

Overexpression of Smad7 or Smad6 Transgene in Embryonic
Mouse Lungs in Culture-To evaluate the biological function of inhibitory Smads during early lung development, we began our study by assessing the AdSmad7 and AdSmad6 for their ability to overexpress the corresponding transgenes in embryonic mouse lungs in culture. E11 mouse lungs were cultured for 4 days as described under "Materials and Methods" following intratracheal micro-injection of AdSmad7, AdSmad6, or a control virus that contains no exogenous genes. Cultured lung explants were harvested for RNA extraction, reverse transcription, and subsequent competitive PCR quantification of specific mRNA amounts. As shown in competitive PCR electrophoretic patterns ( Fig. 2A), only lungs micro-injected with AdSmad7, not with control virus, displayed an elevated level of Smad7 mRNA transgene expression. However, endogenous mRNA levels of both Smad6 and Smad3 were not changed by AdSmad7 infection into embryonic lungs. Likewise, AdSmad6 infection increased Smad6 mRNA expression in cultured lungs in comparison to control virus-infected lung explants, whereas both endogenous mRNA amounts of Smad7 and Smad3 were unaffected ( Fig. 2A). Quantitative analysis following densitometric scanning of competitive PCR electrophoretic patterns demonstrated a 25-and a 21-fold overexpression (p Ͻ 0.05) of the respective Smad7 and Smad6 transgene mRNAs, when embryonic mouse lungs were micro-injected with either AdSmad7 or AdSmad6 (Fig. 2B).
Significant overexpression of exogenous Smad7 and Smad6 protein was detected in the cultured lungs following administration of respective AdSmad7 and AdSmad6, as shown in Western blot analysis using an anti-FLAG antibody (Fig. 3A). FLAG immunoreactivity was absent in media control lungs. We have previously demonstrated that adenovirus-mediated gene transfer resulted in epithelium-specific transgene expression in cultured mouse lungs (11). Hence, to localize Smad7 or Smad6 transgene expression in embryonic lungs micro-injected with AdSmad7 or AdSmad6, immunohistochemistry was performed on cultured lung specimens using an anti-FLAG polyclonal antibody (Fig. 3B). The staining of exogenous transgene protein was exclusively found in the lung epithelial cells in either AdSmad7 or AdSmad6 micro-injected lungs (Fig. 3B, a for AdSmad7 and b for AdSmad6), whereas mesenchymal cells were free of transgene protein expression. The FLAG staining was also absent in both media control (Fig. 3B, c) and control virus-injected (not shown) lung tissues. Immunocytochemistry using control goat IgG yielded negligible background staining (data not shown).
We thus established an experimental system to successfully overexpress Smad7 and Smad6 genes, at both mRNA and protein levels, using intratracheal micro-injection of respective AdSmad7 and AdSmad6, in embryonic mouse lungs in culture.
Expression of Exogenous Smad7, but Not Smad6, Abrogates TGF-␤1-mediated Inhibition of Embryonic Lung Branching Morphogenesis-By having demonstrated the ability of the AdSmad7 or AdSmad6 to overexpress recombinant Smad7 or Smad6 transgene in the embryonic lungs in culture, we sought to determine whether overexpression of the exogenous inhibi-tory Smad gene could regulate TGF-␤-induced lung branching inhibition in embryonic mouse lungs in culture.
Embryonic murine lungs (E11) spontaneously underwent extensive morphogenesis in chemically defined, serum-free culture to develop into a characteristic branching pattern (Fig. 4A,  a and b). As we have previously shown (10), TGF-␤ ligands inhibited lung branching morphogenesis in culture as quantified by the number of terminal sacs, resulting in a hypoplastic phenotype. In the current study, addition of TGF-␤1 ligand (10 ng/ml) decreased embryonic mouse lung branching morphogenesis in culture in lungs micro-injected with the control virus FIG. 2. Quantitative analysis of mRNA expression of Smad7 or Smad6 transgene in AdSmad7 or AdSmad6-infected lungs after culture. Smad7 or Smad6 mRNA levels were measured by competitive reverse transcriptase-PCR in total RNA prepared from cultured lung explants. A, as shown by competitive PCR electrophoretic patterns, lungs micro-injected with AdSmad7 resulted in mouse Smad7 mRNA overexpression, in comparison to lungs with control virus (CV) microinjection. Both Smad6 and Smad3 gene expression were not altered in AdSmad7-treated lungs. Similarly, lungs micro-injected with AdSmad6 showed an increased mouse Smad6 mRNA over control virus microinjected lungs, whereas mRNA amounts of both Smad7 and Smad3 were unchanged regardless of AdSmad6 infection. B, measurement of endogenous Smad7 or Smad6 mRNA levels in AdSmad7 or AdSmad6 micro-injected lungs after densitometric analysis of intensities of bands shown in A. Micro-injection of AdSmad7 or AdSmad6 significantly (p Ͻ 0.05) increased corresponding endogenous mouse Smad7 or Smad6 mRNA expression in cultured embryonic lungs, whereas both control virus micro-injected and medium control (MC) lungs yielded basal levels of Smad7 or Smad6 expression. Error bars represent S.D. (Fig. 4A, c and d). However, exogenously added TGF-␤1 failed to inhibit lung branching in AdSmad7 micro-injected lungs (Fig. 4A, e), in comparison to control virus micro-injected lungs (d). In contrast, embryonic lungs overexpressing Smad6 could not prevent TGF-␤1-mediated inhibition of lung branching morphogenesis (Fig. 4A, f).
Quantitative analysis of lung branching morphogenesis showed that Smad7, but not Smad6, could reverse TGF-␤mediated inhibition of lung growth in culture. As shown in Fig.  4B, whereas addition of TGF-␤1 ligand inhibited lung branching in either media control or control virus-infected lungs, embryonic lungs overexpressing Smad7 using AdSmad7 microinjection prevented TGF-␤1-mediated lung branching inhibition. In lungs micro-injected with AdSmad7, TGF-␤1 at a dose of 10 ng/ml did not reduce lung branching (103% of media control), whereas TGF-␤1 of the same dose significantly decreased lung morphogenesis in either media control (65% of media control, p Ͻ 0.05) or control virus-treated (68% of media control, p Ͻ 0.05) lungs. However, the above resistance to TGF-␤1-induced lung branching inhibition was not observed when AdSmad6 replaced AdSmad7 for micro-injection into embryonic mouse lungs; TGF-␤1 exerted an inhibitory effect on lung morphogenesis regardless of the micro-injection of AdS-mad6 (79% of media control, p Ͻ 0.05). Our findings therefore demonstrated that only Smad7, but not Smad6, overexpression in lung epithelium rendered embryonic lungs refractoriness to the TGF-␤-mediated negative regulation of lung branching morphogenesis in culture.

␤1-treated
Lungs in Culture-To analyze further the molecular mechanism by which Smad7 transgene expression abolished the TGF-␤-mediated inhibitory effect on pulmonary morphogenesis during early lung development, embryonic mouse lungs were cultured in the presence of TGF-␤ ligand following microinjection of control virus, AdSmad7, or AdSmad6. Phosphorylation of Smad2 by the activated TGF-␤1 type I receptor kinase has been shown to relay the transduction of TGF-␤1 signal from cell membrane to nucleus (18,23). Smad7 associates stably with the activated TGF-␤ receptor complex and interferes with the activation of intracellular Smad2 by preventing its receptor binding and phosphorylation, thereby antagonizing TGF-␤ signaling (17,18). Thus, we examined whether expression of Smad7 or Smad6 transgene could impact on the phosphorylation of TGF-␤ signaling permissive Smad2 in the cultured embryonic lungs. By using an antibody against phosphorylated Smad2 in Western blot analysis, phosphorylation level of Smad2 was found to be increased with the addition of TGF-␤1 ligand in media control lungs (Fig. 5). However, such a TGF-␤1-induced up-regulation of Smad2 phosphorylation was attenuated in FIG. 3. Smad7 or Smad6 transgene protein expression in cultured embryonic lungs after AdSmad7 or AdSmad6 micro-injection. A, Western blotting for cultured lungs using an anti-FLAG monoclonal antibody. Immunoreactive FLAG was only detected in AdSmad7or AdSmad6-infected lungs, and media control (MC) lungs did not show any FLAG immunoactivity. B, cellular localization of Smad7 or Smad6 transgene protein in the cultured embryonic mouse lungs after AdS-mad7 or AdSmad6 treatment. Immunohistochemical staining using an anti-FLAG antibody was performed to visualize transgene expression in cultured lungs micro-injected with AdSmad7 (a), AdSmad6 (b), or control virus (c). Immunostaining was mainly observed in the airway epithelial cells in either AdSmad7 or AdSmad6-infected lungs, whereas lungs treated with control virus were free of staining. Bar, 25 m.

FIG. 4. Effects of exogenous Smad7 or Smad6 on lung branching morphogenesis in culture.
A, embryonic lung branching morphogenesis in culture. E11 lung (a) underwent branching morphogenesis in serumless culture to develop into a characteristic branching pattern (b) after 4 days. Exogenously added TGF-␤1 (d) inhibited lung branching morphogenesis in lungs micro-injected with control virus (c, represent a control virus-infected lung without TGF-␤1 addition). Addition of TGF-␤1 failed to reduce lung branching in lungs micro-injected with AdSmad7 (e), whereas TGF-␤1 showed an inhibitory effect on lung branching regardless of the AdSmad6 micro-injection (f). Bar indicates 100 m. B, quantification of lung branching morphogenesis after culture. Whereas TGF-␤1 inhibited lung branching morphogenesis in both media control (MC) and control virus-infected lungs, intratracheal micro-injection of AdSmad7 prevented the TGF-␤1-induced inhibitory effect on lung branching in culture (p Ͻ 0.05). However, overexpression of Smad6 did not reverse TGF-␤1-mediated inhibition of lung morphogenesis. Error bars indicate S.D.
AdSmad7-infected lungs overexpressing Smad7, regardless of the presence of TGF-␤1 in culture, whereas Smad6 gene transfer into lungs did not affect the stimulatory effect of TGF-␤1 on Smad2 phosphorylation in the mouse lungs culture (Fig. 5). The above observation that the expression of exogenous Smad7, but not Smad6, blocked TGF-␤1-activated Smad2 phosphorylation in cultured embryonic lungs indicates that only Smad7 overexpression could antagonize TGF-␤ signaling during early lung branching morphogenesis. Therefore, Smad7 and Smad6 seem to exhibit differential regulatory effects on TGF-␤ signaling during early lung branching morphogenesis.

TGF-␤1-induced Inhibition on SP-C Gene Expression Is Abolished in Cultured Mouse
Lungs Overexpressing Smad7-The biological function of inhibitory Smads was also examined for their role on pulmonary cytodifferentiation during early lung morphogenesis. Gene expression of surfactant proteins is well characterized to be inhibited by TGF-␤ signaling during embryonic lung development. Since surfactant proteins were primarily expressed in lung epithelium during embryonic lung growth, SP-C was used as a lung epithelium-specific differentiation marker gene to evaluate the role of Smad7 versus Smad6 during early lung growth and cytodifferentiation. When added exogenously to the culture medium, TGF-␤1 repressed epithelial SP-C gene expression in media control lungs (data not shown) and in lungs infected with control virus (Fig. 6), as shown by the competitive PCR electrophoretic pattern for SP-C mRNA measurement. However, exogenous TGF-␤1 no longer down-regulated epithelial SP-C mRNA expression when lungs were epithelially infected with AdSmad7. In comparison, epithelium-specific overexpression of Smad6 failed to reverse TGF-␤1-induced negative regulation of SP-C gene expression in culture embryonic lungs (Fig. 6). Therefore, only Smad7, but not Smad6, transgene expression prevents the embryonic lungs in culture from TGF-␤-mediated inhibition of both lung branching morphogenesis and epithelial differentiation. DISCUSSION In the present study, we showed that transient gene transfer and expression of Smad7, but not Smad6, introduced by intratracheal micro-injection of replication-defective recombinant adenoviral vector into the embryonic mouse lungs in culture, prevented TGF-␤-mediated inhibition of both lung growth and cytodifferentiation, as demonstrated by lung terminal branching morphogenesis and gene expression of epithelial SP-C. Moreover, we have demonstrated that the overexpression of Smad7 alone in lung epithelium is sufficient to block the TGF-␤-mediated inhibition of lung epithelial branching and differentiation. We also provided direct evidence that exogenous Smad7 may exert its anti-TGF-␤ signaling effect in cultured embryonic lungs by the blockade of Smad2 phosphorylation, the pivotal step for the transduction of TGF-␤ signal from extracellular membrane to nucleus. Since the binding of Smad2 to activated TGF-␤ type I receptor is required for the Smad2 phosphorylation, it is likely that Smad7 antagonizes TGF-␤ signal transduction through interfering with the association of Smad2 to its cognate receptor complex during lung morphogenesis.
Vertebrate Smad6 and Smad7 and Drosophila Dad are inhibitors of signaling by receptor-activated Smads (17,18,22,24,25). When overexpressed, Smad6 can inhibit BMP signaling, and Smad7 can inhibit TGF-␤ superfamily signaling (17,18,22). Dad inhibits Dpp signaling in Drosophila wing imaginal discs, and when introduced into frog embryos, Dad exhibits anti-BMP effects (25). Inhibitory Smads participate in negative feedback loops that may regulate the intensity or duration of TGF-␤ responses. Thus, Smad7 expression is rapidly elevated in response to TGF-␤, whereas Dad expression is increased in response to Dpp (18,25). However, the biological function of Smad7 during early embryogenesis has not been defined yet. Our findings suggested that Smad7 plays a functional role as an antagonist of TGF-␤ signaling during embryonic mouse lung branching morphogenesis and cytodifferentiation.
Although both Smad7 and Smad6 are implicated in the negative modulation of the TGF-␤ superfamily signaling, we found, in the current investigation, that Smad7 and Smad6 displayed differential inhibitory effects on both lung morphogenesis and epithelial cell differentiation. In comparison to Smad7, Smad6 did not affect TGF-␤-mediated negative regulation of lung growth and development in mice. Although Smad6 was originally identified as an inhibitor of signaling by the TGF-␤ superfamily (22), recent studies indicate that Smad6 preferentially attenuates signaling of BMPs (26,27). Smad6 has been shown to bind to BMP receptors, inhibit signaling downstream of the BMP receptors (22), and prevent formation of an active Smad1/Smad4 signaling complex by directly competing with Smad1 for binding to Smad4 (27). Mouse Smad6 promoter was identified to contain BMP-responsive elements, and the expression of Smad6 is enhanced by the effects of BMP-activated Smad1/5 on the Smad6 promoter (28). Therefore, the above findings are consistent with our observation in lung culture that TGF-␤ signaling activity is inert to the regulation by Smad6 gene.
Signaling by peptide growth factors plays a critical role in lung morphogenesis, determining the rate of cell proliferation and differentiation, activation or repression of downstream  6. SP-C mRNA expression in AdSmad7/AdSmad6 microinjected lungs. Cultured lungs were extracted for total RNA and subsequently subjected to competitive reverse transcriptase-PCR assays. As shown in competitive PCR electrophoretic pattern, TGF-␤1 reduced SP-C mRNA level in control virus-treated lungs in culture. Such a TGF-␤1-mediated suppression of SP-C mRNA expression was abolished in cultured lungs micro-injected with AdSmad7 but not AdSmad6.
transcriptional factors, and synthesis of extracellular matrix proteins. TGF-␤ is a known negative regulator in developing lungs. TGF-␤ ligands have been shown to exert an inhibitory effect on lung branching morphogenesis and simultaneously suppress surfactant protein gene expression in pulmonary epithelium, in both in vivo animal models and ex vivo lung organ cultures (7,9,10). Our previous study demonstrated that abrogation of endogenous Smad2 and Smad3 using antisense oligodeoxynucleotides results in a stimulatory phenotype for branching morphogenesis of early murine embryonic lungs in culture (29), similar to that obtained after abrogation of TGF-␤ type II receptor signaling (10). In the present report, we show that overexpression of Smad7 prevents exogenous TGF-␤-induced Smad2 phosphorylation in developing lungs in culture, indicating a lack of association between Smad2 and TGF-␤ type I receptor in the presence of Smad7. Taken together, it is suggested that Smad7 exerts its biological effects on both lung branching and differentiation through attenuation of TGF-␤ signal transduction, probably by competing with Smad2 for TGF-␤ receptor binding.
Elevated TGF-␤ signaling appears to be a characteristic feature of fibrotic diseases including hepatic cirrhosis, pulmonary fibrosis, and glomerular sclerosis (30). Aberrant expression of TGF-␤ signaling, occurring during lung disease and injury, could perturb finely regulated TGF-␤-mediated molecular cascades, resulting in abnormalities of lung growth, differentiation, and development (12). Expression of Smad7 ameliorated bleomycininduced lung fibrosis in mice by preventing the increased production of type I pre-collagen mRNA and hydroxyproline content (31). By using embryonic mouse lung explant culture, we found that Smad7 gene transfer attenuated TGF-␤ signaling in developing lungs, reversing TGF-␤-mediated inhibition of both epithelial branching and SP-C gene expression. Therefore, Smad7 expression in the lung offers a novel rationale for therapeutic intervention that may be useful in treating lung diseases associated with TGF-␤ overproduction.
Smad7 itself is induced by TGF-␤ stimulation, suggesting that Smad7 regulates TGF-␤ signaling in a negative feedback fashion (4,32). However, recent studies have revealed that Smad7 may be activated by other signaling pathways. TGF-␤ and interferon-␥ have opposite effects on diverse cellular functions (33). Acting through Jak1 and Stat1, interferon-␥ induces the expression of Smad7, which prevents TGF-␤ receptor from interaction with and phosphorylation of the intracellular Smad3 (34). Likewise, tumor necrosis factor-␣/NF-B was found to antagonize TGF-␤ signaling through up-regulation of Smad7 synthesis and induction of stable associations between ligand-activated TGF-␤ receptors and inhibitory Smad7 (35). Moreover, recent experimental evidences have demonstrated that permissive Smads synergize with members of other transcription factor families including FAST-1, -2, AP-1, and p300, in the regulation of downstream gene expression, indicating the versatility of Smad signaling system (36). Therefore, Smad7 may not only be responsive to TGF-␤ signaling but also may act as a potential signaling integrator that could play a key role in the interplay of different signaling pathways. However, the precise role of Smad7 as a signal integrator during lung development remains to be explored.
In the current analysis, we used an adenovector-mediated gene transfer strategy to achieve overexpression of Smad7 during embryonic lung morphogenesis. Intratracheal micro-injection of recombinant adenovirus to introduce transgene expression has proven to be both effective and efficient (11). The replication-deficient adenovirus has a wide spectrum of host cell range, including cells in both active and quiescent states (37). The recombinant adenoviruses used herein are incapable of integrating into the host genome and thus have minimum impact on intrinsic host cell function. By using both Smad6 and Smad7 adenoviruses, we have shown the feasibility of achieving high transgene expression specifically in lung epithelial cells during embryonic lung growth and development. Our data therefore support the hypothesis that Smad7, as an intracellular antagonist of TGF-␤ pathway, may be potentially applicable as a target in designing novel strategies to treat lung diseases and injuries due to excessive TGF-␤ signaling.
In summary, we have demonstrated that transient gene transfer and expression of exogenous Smad7 and Smad6 into the lung epithelium by adenovectors differentially modulated TGF-␤-induced inhibition of both lung branching morphogenesis and epithelial cell differentiation. Smad7, but not Smad6, may antagonize TGF-␤ signaling in the developing lungs though blockade of TGF-␤ receptor-activated Smad2 phosphorylation. Smad7 thus appears to be an important biological regulator that optimizes TGF-␤ signaling during lung growth and development, and Smad7 could be a potentially useful molecular target in designing new rational therapeutic strategies to ameliorate lung injuries due to abnormal TGF-␤ signal transduction.