Daughters against dpp
Smad6 has been isolated in the mouse. Smad6 is quite different in structure from the other SMAD proteins, and forms stable associations with type I receptors. Smad6 interferes with the phosphorylation of Smad2 and the subsequent heteromerization with Smad4, but does not inhibit the activity of Smad3. Smad6 also inhibits the phosphorylation of Smad1 that is induced by the bone morphogenetic protein type IB receptor. These data indicate that signals of the TGF-beta superfamily are regulated both positively and negatively by members of the SMAD family (Imamura, 1997).
Identification of Smad7, which is related to Smad6, has been reported. Transfection of Smad7 blocks responses mediated by TGF-beta in mammalian cells, and injection of Smad7 RNA into Xenopus embryos blocks activin/TGF-beta signaling. Smad7 associates stably with the TGF-beta receptor complex, but is not phosphorylated upon TGF-beta stimulation. TGFbeta-mediated phosphorylation of Smad2 and Smad3 is inhibited by Smad7, indicating that the antagonistic effect of Smad7 is exerted at this important regulatory step. TGF-beta rapidly induces expression of Smad7 mRNA, suggesting that Smad7 may participate in a negative feedback loop to control TGF-beta responses (Nakao, 1997).
Smad6 and Smad7 comprise a subclass of vertebrate Smads that antagonize, rather than transduce, TGF-beta family signaling. These Anti-Smads can block BMP signaling, as evidenced by their ability to induce a secondary dorsal axis when misexpressed ventrally in Xenopus embryos. Smad7 inhibits additional TGF-beta related pathways, and causes spina bifida when misexpressed dorsally. Structure-function analyses have been performed to identify domains of Anti-Smads that are responsible for their shared and unique activities. The C-terminal domain of Smad7 displays strong axis inducing activity but cannot induce spina bifida. The isolated N-terminal domain of Smad7 is inactive but restores the ability of the C-terminus to cause spina bifida when the two are co-expressed. By contrast, the N- and C-terminal domains of Smad6 have weak axis inducing activity when expressed individually, but show full activity when co-expressed. Chimeric analysis demonstrates that the C-terminal domain of Smad7, but not Smad6, can induce spina bifida when fused to the N-terminal domain of either Smad6 or Smad7. Thus, although the C-terminal domain is the primary determinant of the intrinsic activity of Xenopus Anti-Smads, the N-terminal domain is essential for full activity, is interchangeable between Smad6 and 7, and can function in trans (Nakayama, 2001).
A variety of evidence suggests that the C-terminal domain of Anti-Smads is an effector domain, whereas the N-domain has an inhibitory effect and/or confers specificity for distinct signaling pathways. The isolated C-domain of human Smad6, for example, binds constitutively to Smad1 and is sufficient to inhibit BMP signal transduction, whereas full length Smad6 binds to Smad1 only when the BMP pathway has been activated. Thus, deletion of the N-terminal domain of Smad6 relieves the ligand dependence of signal inhibition. The C-terminal domain of Smad6 is also sufficient for binding to Hoxc-8, thereby preventing Smad1 from interacting with Hoxc-8 and activating transcription. Interaction of full length Smad6 with Hoxc-8 is much weaker, again consistent with an inhibitory role for the N-domain. Likewise, an intact C-terminal domain of Smad7 has been shown to be necessary and sufficient for association with TGF-beta family receptors and signal inhibition. Recent studies have also shown that an intact C-terminal domain of Smad7 is required for interaction with STRAP, a protein that stabilizes the interaction of Smad7 with the intracellular domain of TGF-beta receptors and thus potentiates its inhibitory effects. Finally, the observation that Xenopus and murine Smad7, which share 96% identity within the C-terminal domain and much less identity within the N-terminal domain, show different biological activities in Xenopus embryo overexpression assays has been interpreted to suggest that the C-terminal domain is an effector whereas sequences within the N-terminal domain confer specificity for distinct signaling pathways (Nakayama, 2001 and references therein).
The current data support the idea that the C-domain of Anti-Smads is primarily responsible for biological activity. The N-domain does not function, however, as an inhibitor and/or a determinant of signaling specificity, but is instead essential for full activity. An N-terminal domain is required, for example, for the C-terminal domain of Smad7 to induce spina bifda or for the C-terminal domain of Smad6 to inhibit BMP signaling. The latter observation is curious given that a comparable or smaller C-terminal domain of Xenopus Smad7, murine Smad7, and human Smad6 is sufficient for inhibition of BMP signaling in Xenopus embryos. The observation that addition of the N-terminal domain of Smad6 can restore full activity to the C-terminal domain of Smad7, and vice versa, clearly demonstrates that the specificity of a given Anti-Smad for a particular signaling pathway is intrinsic to the C-terminal domain. In addition to being required for full C-domain function, it was found that the isolated N-domain of Smad6 is itself functional. This finding is unexpected and raises the possibility that the recently reported targeted disruption of the murine Smad6 gene is not a true null allele since only the MH2 domain is deleted (Nakayama, 2001 and references therein).
The molecular mechanism by which the N-terminal domain contributes to the activity of Anti-Smads is unknown but several possibilities can be imagined. It is possible that the N-domain interacts with the C-domain to establish an active conformation that is required for some Anti-Smad functions. If this is the case, these two domains must physically interact even when non-covalently attached, since the N- and C-domains can function in trans when co-expressed. Consistent with this possibility, the MH1 domains of Smad2 and 4 can directly bind to their respective MH2 domains. Attempts have been made to co-immunoprecipitate Smad6 N and Smad6 DN and no evidence has been found that these domains interact either in vitro or in vivo (Nakayama, 2001).
An alternate mechanism by which the N-domain of Anti- Smads might function is to recruit a co-factor that is essential for some Anti-Smad functions. Conversely, the N-domain might bind to, and thus prevent an inhibitory factor from interacting with the C-terminal domain in vivo. This latter possibility might account for the observation that the isolated N-domain of Smad6 has biological activity in overexpression assays, since it could conceivably displace an inhibitory protein bound to the C-terminus of endogenous Smad6 and thereby unmask its intrinsic activity. This possibility might also explain the ability of the isolated C-terminal domain of mammalian, but not Xenopus, Smad6 to inhibit BMP signaling in Xenopus embryos if this hypothetical binding protein can only recognize the Xenopus C-terminus due to species specifc sequence differences (Nakayama, 2001).
Specific sequence elements (amino acids 115-153) must be covalently attached to the N-terminal region of Smad6 to generate a functional domain. Furthermore, amino acids 161-171 of Smad7 are essential for its ability to induce spina bifda. The functional N-termini of Smad6 and Smad7 contain several highly conserved sequence motifs that are found only in Anti-Smads (consensus AVESRGG, corresponding to amino acids 61-67 and 81-87 of Xenopus Smad6 and Smad7, and consensus ESPPPPYSR, corresponding to amino acids 134-142 and 161-169 of Xenopus Smad6 and Smad7, respectively). These motifs may act as important determinants of N-domain function, possibly by creating binding pockets for accessory proteins. The conserved ESPPPPYSR sequence, for example, contains a PPPY motif that is a possible binding site for WW domain-containing proteins. Notably, the SMAD ubiquitin ligase, Smurf1, is a WW domain-containing protein that binds to a PPXY motif in the R-Smad, Smad1, thereby targeting it for degradation by the 26S proteosome. The observation that deletion of a region of Smad6 or Smad7 encompassing this motif reduces or ablates activity raises the possibility that the function of Anti-Smads may be regulated by a WW domain-containing protein(s). Further identification of binding proteins that modulate the activities of Anti-Smads will help to clarify these issues. Regardless of the mechanism of action of the N-terminal domain, the data show that current models for Anti-Smad function, in which the C-terminal domain binds to TGF-beta family receptors, signal transducing Smads, or transcription factors are not adequate to explain the full in vivo activities of these molecules (Nakayama, 2001).
TGFbeta signaling is initiated when the type I receptor phosphorylates the MAD-related protein, Smad2, on C-terminal serine residues. This leads to Smad2 association with Smad4, translocation to the nucleus, and regulation of transcriptional responses. Smad7 is an inhibitor of TGFbeta signaling. Smad7 prevents TGFbeta-dependent formation of Smad2/Smad4 complexes and inhibits the nuclear accumulation of Smad2. Smad7 interacts stably with the activated TGFbeta type I receptor, thereby blocking the association, phosphorylation, and activation of Smad2. Furthermore, mutations in Smad7 that interfere with receptor binding disrupt its inhibitory activity. These studies thus define a novel function for MAD-related proteins as intracellular antagonists of the type I kinase domain of TGFbeta family receptors (Hayashi, 1997).
A WD40 repeat protein, STRAP, associates with both type I and type II TGF-beta receptors and is involved in TGF-beta signaling. STRAP synergizes specifically with Smad7, but not with Smad6, in the inhibition of TGF-beta-induced transcriptional responses. STRAP does not show cooperation with a C-terminal deletion mutant of Smad7 that does not bind with the receptor and consequently has no inhibitory activity. STRAP associates stably with Smad7, but not with the Smad7 mutant. STRAP recruits Smad7 to the activated type I receptor and forms a complex. Moreover, STRAP stabilizes the association between Smad7 and the activated receptor, thus assisting Smad7 in preventing Smad2 and Smad3 access to the receptor. STRAP interacts with Smad2 and Smad3 but does not cooperate functionally with these Smads to transactivate TGF-beta-dependent transcription. The C terminus of STRAP is required for its phosphorylation in vivo; this is dependent on the TGF-beta receptor kinases. Thus, a mechanism is described to explain how STRAP and Smad7 function synergistically to block TGF-beta-induced transcriptional activation (Datta, 2000).
Bone morphogenetic protein 2 (BMP2), a member of the transforming growth factor-beta (TGF-beta) superfamily, regulates a variety of cell fates and functions. At present, the molecular mechanism by which BMP2 induces apoptosis has not been fully elucidated. A BMP2 signaling pathway is proposed that mediates apoptosis in mouse hybridoma MH60 cells [whose growth is interleukin-6 (IL-6)-dependent]. BMP2 dose-dependently induces apoptosis in MH60 cells, even in the presence of IL-6. BMP2 has no inhibitory effect on the IL-6-induced tyrosine phosphorylation of STAT3; the bcl-2 gene expression, which is known to be regulated by STAT3, suggests that BMP2-induced apoptosis is not attributed to alteration of the IL-6-mediated bcl-2 pathway. BMP2 induces activation of TGF-beta-activated kinase (TAK1) and subsequent phosphorylation of p38 stress-activated protein kinase. In addition, forced expression of kinase-negative TAK1 in MH60 cells blocks BMP2-induced apoptosis. These results indicate that BMP2-induced apoptosis is mediated through the TAK1-p38 pathway in MH60 cells. MH60-derived transfectants expressing Smad6 are resistant to the apoptotic signal of BMP2. Interestingly, this ectopic expression of Smad6 blocks BMP2-induced TAK1 activation and p38 phosphorylation. Moreover, Smad6 can directly bind to TAK1. These findings suggest that Smad6 is likely to function as a negative regulator of the TAK1 pathway in the BMP2 signaling, in addition to the previously reported Smad pathway (Kimura, 2000).
Smad6 and Smad7 function as intracellular antagonists in transforming growth factor-beta (TGF-beta) signaling. Human Smad6 is closely related to Smad7. Smad6 and Smad7 mRNAs are differentially expressed in lung cancer cell lines and are rapidly and directly induced by TGF-beta1, activin and bone morphogenetic protein-7. Cross-talk between TGF-beta and other signaling pathways has been demonstrated by the finding that epidermal growth factor (EGF) induces the expression of inhibitory SMAD mRNA. Moreover, whereas the phorbol ester PMA alone has no effect, it potentiates the TGF-beta1-induced expression of Smad7 mRNA. Ectopic expression of anti-sense Smad7 RNA increases the effect of TGF-beta1, supporting its role as a negative regulator in TGF-beta signaling. Thus, expression of inhibitory Smads is induced by multiple stimuli, including the various TGF-beta family members, whose actions they antagonize (Afrakhte, 1998).
Expression of the Smad6 mRNA is dramatically induced by BMP-2 or osteogenic protein-1 (OP-1)/BMP-7 in various cells. BMP-2 induces expression of Smad7 in one cell type, although much less potently than that of Smad6. Smad6 message is induced by TGF-beta 1 in TGF-beta 1-responsive Mv1Lu cells, but the induction is transient in contrast to the induction by BMPs. These results indicate that Smad6 may form a feedback loop to regulate the signaling activity of BMPs (Takase, 1998).
The detailed characterization of the rat Smad7 genomic organization including the promoter region is presented. The gene is composed of four exons separated by three introns covering a DNA region of about 30 kilobases (kb) in total. The major transcription start site is conserved between rat and mouse, and two polyadenylation signals have been detected. In the promoter region, a potential CAGA box, a signal transducer and activator of transcription (STAT) factor-related recognition site, and different AP1 sites have been identified, which could be the targets of TGFbeta, IFN-gamma, and EGF-dependent Smad7 transcription initiation (Stopa, 2000).
Smad6 is an inhibitory Smad that is induced by bone morphogenetic proteins (BMPs) and interferes with BMP signaling. The mouse Smad6 promoter has been isolated and the regions responsible for transcriptional activation by BMPs have been identified. The proximal BMP-responsive element (PBE) in the Smad6 promoter is important for the transcriptional activation by BMPs and contains a 28-base pair GC-rich sequence, including four overlapping copies of the GCCGnCGC-like motif, which is a binding site for Drosophila Mad and Medea. A luciferase reporter construct (3GC2-Lux), containing three repeats of the GC-rich sequence derived from the PBE, has been generated. BMPs and BMP receptors induce transcriptional activation of 3GC2-Lux in various cell types, and this activation is enhanced by cotransfection of BMP-responsive Smads, i.e. Smad1 or Smad5. Moreover, direct DNA binding of BMP-responsive Smads and common-partner Smad4 to the GC-rich sequence of PBE is observed. These results indicate that the expression of Smad6 is regulated by the effects of BMP-activated Smad1/5 on the Smad6 promoter (Ishida, 2000).
Smad7, but not Smad6, inhibits TGF-beta1-induced growth inhibition and the expression of immediate early response genes, including Smad7. Interestingly, in the absence of ligand, Smad7 is found predominantly localized in the nucleus, whereas Smad7 accumulates in the cytoplasm upon TGF-beta receptor activation. The latter is in accordance with the physical association of Smad7 with the ligand-activated TGF-beta receptor complex in the cell membrane. Whereas the ectopically expressed C-terminal domain of Smad7 is also exported from the nucleus to the cytoplasm upon TGF-beta challenge, a Smad7 mutant with a small deletion at the C terminus or only the N-terminal domain of Smad7 is localized mainly in the cytoplasm in the absence or presence of ligand. This suggests that an intact Mad homology 2 domain is important for nuclear localization of Smad7. The nuclear localization of Smad7 suggests a functional role distinct from its antagonistic effect in receptor-mediated Smad activation (Itoh, 1998).
Smad6 and Smad7, a subgroup of Smad proteins, antagonize the signals elicited by transforming growth factor-beta. These two Smads, induced by transforming growth factor-beta or bone morphogenetic protein (BMP) stimulation, form stable associations with their activated type I receptors, blocking phosphorylation of receptor-regulated Smads in the cytoplasm. Smad6 interacts with homeobox (Hox) c-8 as a transcriptional corepressor, inhibiting BMP signaling in the nucleus. The interaction between Smad6 and Hoxc-8 was identified by a yeast two-hybrid approach and further demonstrated by co-immunoprecipitation assays in cells. Gel shift assays show that Smad6, but not Smad7, interacts as a heterodimer with both Hoxc-8 and Hoxa-9 when binding to DNA. More importantly, the Smad6-Hoxc-8 complex inhibits interaction of Smad1 with Hoxc-8- and Smad1-induced transcription activity. These data indicate that Smad6 interacts with Hox transcription factors as part of the negative feedback circuit in the BMP signaling pathway (Bai, 2000).
A new inhibitory Smad in Xenopus has been isolated and characterized. Smad7 is present at fairly constant levels throughout early development and at blastula stages enriched in the ventral side of the animal hemisphere. The induction of mesoderm by TGF-beta-like signals is mediated by receptor ALK-4 and Smad7 has been shown to block signaling of ALK-4 in a graded fashion: lower levels of Smad7 block activation of dorsal mesoderm genes and higher levels block all mesoderm genes expression. Smad7 is able to directly activate neural markers in explants in the absence of mesoderm or endoderm. This neural-inducing activity of Smad7 may be due to inhibition of BMP-4 signaling because Smad7 can also block BMP-4-mediated mesoderm induction. Thus, Smad7 acts as a potent inhibitor of mesoderm formation and also activates the default neural induction pathway (Bhushan, 1998).
Smads are proteins that transduce signals on behalf of members of the TGF beta superfamily of growth factors. Recently, three inhibitory Smads (Smad6, Smad7, and Dad) were isolated from human, mouse, and fly respectively. These anti-Smads have been shown to inhibit TGF beta signaling by stably associating to TGF beta type I receptors or, as was shown for Smad6, by binding to receptor-activated Smad1. Xenopus Smad7 (XSmad7) inhibits signaling from the activin and BMP pathways in animal explants, although at different thresholds. When expressed in the embryo, low concentrations of XSmad7 dorsalize the ventral mesoderm, thus inducing a secondary axis. At higher concentrations however, XSmad7 inhibits both mesoderm induction and primary axis specification. In addition, XSmad7 acts as a direct neural inducer both in the context of ectodermal explants and in vivo. It is suggested that XSmad7 affects distinct TGFbeta pathways at different thresholds: at low doses, it selectively blocks the BMP pathway, whereas at higher concentrations, it is additionally capable of inhibiting the activin/TGFbeta-like pathways. XSmad7 is present maternally and maintains ubiquitous expression at least until the onset of gastrulation when the mesoderm, ectoderm, and endodem are specified (Casellas, 1998).
Bone morphogenetic proteins (BMPs) transmit signals via the intracellular protein Smad1, which is phosphorylated by ligand bound receptors, translocates to the nucleus, and functions to activate BMP target genes. Recently, a subclass of Smad proteins has been shown to inhibit, rather than transduce, BMP signaling, either by binding to the intracellular domain of BMP receptors, thereby preventing phosphorylation-mediated activation of Smad1, or by binding directly to Smad1, thereby inhibiting its ability to activate gene transcription. A Xenopus Smad (Smad6) has been identified that is 52% identical to mammalian Smad6, an inhibitory Smad. The spatial pattern of expression of Smad6 changes dynamically during embryogenesis and is similar to that of BMP-4 at the tailbud stage. Overexpression of Smad6 in Xenopus embryos phenocopies the effect of blocking BMP-4 signaling, leading to dorsalization of mesoderm and neuralization of ectoderm. Xenopus Smad6 completely blocks the activity of exogenous BMP-4, and, unlike human Smad6, partially blocks the activity of activin, in a mesoderm induction assay. Smad6 protein accumulates at the membrane in some cells but is partially or completely restricted to nuclei of most overexpressing cells. Thus Smad6 functions as an intracellular antagonist of activin and BMP-4 signaling. The finding that Smad6 protein is partially or completely restricted to the nuclei of most overexpressing cells suggests that it may employ a novel or additional mechanism of action to antagonize TGF-beta family signaling, other than that reported for other inhibitory Smads (Nakayama, 1998).
Smad6 and Smad7 prevent ligand-induced activation of signal-transducing Smad proteins in the transforming growth factor-beta family. 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. The ectopic expressions of Smad6 and Smad7 inhibit hBMP-2-induced Smad1/Smad5 phosphorylation. 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 is induced by activin A in HS-72 cells, Smad6 shows no antagonistic effect on activin A-induced growth arrest and apoptosis. Moreover, the ectopic expression of Smad7, but not Smad6, inhibits 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 (Ishishki, 1999).
Bone morphogenetic protein (BMP) receptors signal by phosphorylating Smad1, which then associates with Smad4; this complex moves into the nucleus and activates transcription. A natural inhibitor of this process is Smad6. In Xenopus embryos and in mammalian cells, Smad6 specifically blocks signaling by the BMP/Smad1 pathway. Smad6 inhibits BMP/Smad1 signaling without interfering with receptor-mediated phosphorylation of Smad1. Smad6 specifically competes with Smad4 for binding to receptor-activated Smad1, yielding an apparently inactive Smad1-Smad6 complex. Therefore, Smad6 selectively antagonizes BMP-activated Smad1 by acting as a Smad4 decoy. In Xenopus, Smad6 can induce cement gland and neural tissues in ectodermal explants in a cell-autonomous and dose-dependent manner, without inducing mesoderm. Smad6 also inhibits induction of a ventral mesoderm marker by a BMP receptor. Similar effects are observed with Dad, a Drosophila homolog of Smad6. Smad6 does interfere with formation of the primary axis, a process that requires signaling via the activin receptor. When directly challenged by Smad6 in a Xenopus animal cap assay, Smad1, but not Smad2, action is inhibited by Smad6. Smad6 inhibits BMP/Smad1 signaling selectively, without inhibiting Smad2 signaling in Xenopus embryos or TGFbeta and activin effects in mammalian cells. Smad6 may be an intracellular complement to the BMP inhibitory functions of Noggin, Chordin, and/or Follistatin and may play a key role in cell-autonomous determination of cell fate (Hata, 1998).
Expression of chicken Smad6 (cSmad6), a member of inhibitory SMADs, is remarkably restricted to the developing heart, eyes, and limbs. cSmad6 expression is detected in the cardiogenic region of stage 5 embryos and overlaps Nkx2-5 and bmp-2, -4, and -7 expression. Throughout development, cSmad6 is expressed strongly in the heart, primarily in the myocardium, endocardium, and endocardial cushion tissue. Myocardial expression of cSmad6 is stronger in the forming septum, where highly localized expression of bmp-2 and -4 is also observed. Ectopically applied BMP-2 protein induces the expression of cSmad6, a putative negative regulator of BMP-signaling pathway, in anterior medial mesoendoderm of stage 4-5 embryos. In addition, blocking of BMP signaling using Noggin downregulates cSmad6 in cardiogenic tissue. cSmad1, one of the positive mediators of BMP signaling, is also expressed in the cardiogenic region, but is not BMP-2 inducible. These data suggest that cSmad6 has a role in orchestrating BMP-mediated cardiac development. It is proposed that cSmad6 modulates BMP signal by keeping a balance between constitutively expressed pathway-specific cSmad1 and ligand-induced inhibitory cSmad6 in the developing heart (Yamada, 1999).
The role of an inhibitory Smad in vivo has been explored by targeted mutation of Madh6 (which encodes the Smad6 protein). Targeted insertion of a LacZ reporter demonstrates that Smad6 expression is largely restricted to the heart and blood vessels, and that Madh6 mutants have multiple cardiovascular abnormalities. Hyperplasia of the cardiac valves and outflow tract septation defects indicate a function for Smad6 in the regulation of endocardial cushion transformation. The role of Smad6 in the homeostasis of the adult cardiovascular system is indicated by the development of aortic ossification and elevated blood pressure in viable mutants. These defects highlight the importance of Smad6 in the tissue-specific modulation of Tgf-beta superfamily signaling pathways in vivo (Galvin, 2000).
Smad6 and Smad7 are both expressed in human adult vascular endothelial cells, particularly after these cells have been subjected to shear stress. Smad7 mRNA is highly expressed in the developing vascular system of the mouse embryo but is also detectable much earlier in preimplantation embryos and during gastrulation. Overexpression of Smad7 in mouse zygotes inhibits development beyond the 2-cell stage. This confirms earlier conclusions of similar, but complementary, experiments using a dominant negative type II TGFbeta receptor demonstrating that TGFbeta signaling is required for normal preimplantation development (Zwijsen, 2000).
Transforming growth factors beta (TGF-beta) are known negative regulators of lung development, and excessive TGF-beta production has been noted in pulmonary hypoplasia associated with lung fibrosis. Inhibitory Smad7 antagonizes TGF-beta family signaling by interfering with the activation of TGF-beta signal-transducing Smad complexes. To investigate whether Smad7 can regulate TGF-beta-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-beta efficiently reduces epithelial lung branching morphogenesis in control virus-infected lung culture, TGF-beta-induced branching inhibition is abolished after epithelial transfer of the Smad7 gene into lungs in culture. Smad7 also prevents TGF-beta-mediated down-regulation of surfactant protein C gene expression, a marker of bronchial epithelial differentiation, in cultured embryonic lungs. Moreover, Smad7 transgene expression blocks Smad2 phosphorylation induced by exogenous TGF-beta ligand in lung culture, indicating that Smad7 exerts its inhibitory effect on both lung growth and epithelial cell differentiation through modulation of TGF-beta pathway-restricted Smad activity. However, the above anti-TGF-beta signal transduction effects are not observed in cultured embryonic lungs with Smad6 adenoviral gene transfer, suggesting that Smad7 and Smad6 differentially regulate TGF-beta signaling in developing lungs. These data therefore provide direct evidence that Smad7, but not Smad6, prevents TGF-beta-mediated inhibition of both lung branching morphogenesis and cytodifferentiation, establishing the mechanistic basis for Smad7 as a novel target to ameliorate aberrant TGF-beta signaling during lung development, injury, and repair (Zhao, 2000).
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