Regulation Of TGF-beta Signaling Research Articles

Regulation of TGF‐beta signaling by RGS3

Regulator of G protein signaling RGS3 belongs to a family of RGS proteins that contain the homologous RGS domain which binds the alpha subunits of heterotrimeric G proteins and accelerates their GTPase activity. Through this mechanism RGS3 regulates the signaling mediated by a variety of Gq and Gi coupled receptors. In this study, we found that RGS3 interacts with the novel partners, Smad2, Smad3 and Smad4 ‐ the transcription factors that are activated through a transforming growth factor‐beta (TGF‐beta) receptor signaling. This interaction is mediated by Smad's Mad homology domain 2 (MH2) and by the region of RGS3 outside of the RGS domain. Overexpression of RGS3 results in inhibition of Smad‐mediated gene transcription. RGS3 does not affect TGF‐beta ‐ induced Smad phosphorylation, but it prevents heteromerization of Smad3 with Smad4, which is required for Smad's transcriptional activity. Functionally, this translates to inhibition of TGF‐beta – induced myofibroblast differentiation by adenovirus‐mediated overexpression of RGS3. In conclusion, this study identifies a novel, non‐canonical role of RGS3 in regulation of TGF‐beta signaling through its interaction with Smad transcription factors.

Regulation of Smad-Mediated Gene Transcription by RGS3

Regulator of G protein signaling (RGS) proteins are united into a family by the presence of the homologous RGS domain that binds the alpha subunits of heterotrimeric G proteins and accelerates their GTPase activity. A member of this family, RGS3 regulates the signaling mediated by G(q) and G(i) proteins by binding the corresponding Galpha subunits. Here we show that RGS3 interacts with the novel partners Smad2, Smad3, and Smad4-the transcription factors that are activated through a transforming growth factor-beta (TGF-beta) receptor signaling. This interaction is mediated by the region of RGS3 outside of the RGS domain and by Smad's Mad homology 2 domain. Overexpression of RGS3 results in inhibition of Smad-mediated gene transcription. RGS3 does not affect TGF-beta-induced Smad phosphorylation, but it prevents heteromerization of Smad3 with Smad4, which is required for transcriptional activity of Smads. This translates to functional inhibition of TGF-beta-induced myofibroblast differentiation by RGS3. In conclusion, this study identifies a novel, noncanonical role of RGS3 in regulation of TGF-beta signaling through its interaction with Smads and interfering with Smad heteromerization.

The evolutionally conserved activity of Dapper2 in antagonizing TGF‐ß signaling

Dapper1 and Dapper2, two divergent members of the Dapper family, have been suggested to modulate Wnt and TGF-beta/Nodal signaling in Xenopus and zebrafish. To get a better understanding of Dapper function in mammals, we have cloned the mouse ortholog of zebrafish Dapper2, mDpr2 and investigated its function in regulating TGF-beta signaling activity. Here, we showed that, like zebrafish Dapper2, overexpression of mDpr2 inhibited the TGF-beta-induced expression of the Smad-responsive reporters and targeted TGF-beta type I receptor ALK5 for degradation in mammalian cells. Overexpression of mDpr2 in the zebrafish embryos led to a decrease in expression of the mesoderm marker no tail and goosecoid at the shield stage and eye fusion later, implying that mDpr2 may have an intrinsic in vivo activity similar to fish Dapper2 activity. The expression of mDpr2 was detected throughout the epiblast around the onset of gastrulation and in somites, the neural tube and gut at later stages in mouse embryos, implying a role in early embryonic development. Our data indicate that the function of Dpr2 as a negative regulator of the TGF-beta/Nodal signal pathway is evolutionally conserved, at least in part, in fish and mammals.

Expression and function of TβRII-B, a variant of the type II TGF-β receptor, in human chondrocytes

Transforming growth factor-beta (TGF-beta) has profound effects on chondrocyte proliferation and matrix production, and dysregulation of TGF-beta action has been implicated in osteoarthritis. The mechanisms by which the diverse actions of TGF-beta are regulated in chondrocytes are unclear. Although it is well documented that TGF-beta signaling is transduced by types I and II receptors, other TGF-beta receptors may play critical roles by regulating signaling receptor activity. Our objective was to examine the expression of TbetaRII-B, a splice variant of the type II TGF-beta receptor, and to analyze its role in regulating TGF-beta signaling in human chondrocytes. TbetaRII-B expression was examined in human cartilage tissue specimens, human chondrocyte cell lines C28/I2 and tsT/AC62, and human primary chondrocytes by Western blot and reverse-transcriptase-polymerase chain reaction. Ligand binding and heteromerization of TbetaRII-B with other TGF-beta receptors on the cell surface were analyzed by affinity labeling, immunoprecipitation, and two-dimensional SDS-PAGE. Regulation of TGF-beta responses by TbetaRII-B was determined by examining Smad2 phosphorylation, Smad3-specific signaling, transcriptional activity, and type II collagen levels. TbetaRII-B is expressed in normal and osteoarthritic human cartilage. Furthermore, it is a dynamic component of the TGF-beta receptor system in human chondrocytes, forming heteromeric complexes with the types I and II TGF-beta receptors, betaglycan and endoglin. Importantly, overexpression of TbetaRII-B leads to enhanced TGF-beta signaling and responses in chondrocytes. These results suggest that TbetaRII-B may play a key role in the regulation of TGF-beta action in human chondrocytes.

Nuclear Retention of the Tumor Suppressor cPML by the Homeodomain Protein TGIF Restricts TGF-β Signaling

The homeodomain protein TGIF has been implicated in the negative regulation of TGF-beta signaling. In this study, we report an unexpected role of TGIF in the inhibition of Smad2 phosphorylation, which occurs by a mechanism independent of its association with Smad2. This inhibitory function of TGIF is executed in concert with c-Jun, which facilitates the interaction of TGIF with cPML, resulting in the nuclear sequestration of cPML and the disruption of the cPML-SARA complex. Notably, knockdown of TGIF by siRNA caused increased association of cPML with SARA and cytoplasmic accumulation of cPML. Furthermore, c-Jun(-/-) fibroblasts exhibit enhanced association of cPML with SARA. c-Jun(-/-) fibroblasts also lose their sensitivity to TGIF-mediated disruption of the cPML-SARA complex and of nuclear sequestration of cPML. We suggest that the interaction of TGIF with cPML through c-Jun may negatively regulate TGF-beta signaling through controlling the localization of cPML and, consequently, the assembly of the cPML-SARA complex.

Transforming growth factor-β during carcinogenesis: the shift from epithelial to mesenchymal signaling

Transforming growth factor-beta (TGF-beta) activates not only TGF-beta type I receptor (TbetaRI) but also c-Jun N-terminal kinase (JNK), changing unphosphorylated Smad3 to its phosphoisoforms: C-terminally phosphorylated Smad3 (pSmad3C) and linker phosphorylated Smad3 (pSmad3L). While the TbetaRI/pSmad3C pathway inhibits growth of normal epithelial cells, JNK/pSmad3L-mediated signaling is involved in invasion by activated mesenchymal cells. During sporadic human colorectal carcinogenesis, TGF-beta signaling confers a selective advantage on tumor cells by shifting from the TbetaRI/pSmad3C pathway characteristic of mature epithelial cells to the JNK/pSmad3L pathway, which is more characteristic of the state of flux shown by the activated mesenchymal cells. JNK acts as a regulator of TGF-beta signaling by increasing the basal level of pSmad3L available for action in the nuclei of the invasive adenocarcinoma, in the meantime shutting down TGF-beta-dependent nuclear activity of pSmad3C. Loss of epithelial homeostasis and acquisition of a migratory, mesenchymal phenotype are essential for tumor invasion. From the viewpoint of TGF-beta signaling, a key therapeutic aim in cancer would be restoration of the lost tumor suppressor function observed in normal colorectal epithelial cells at the expense of effects promoting aggressive behavior of the adenocarcinoma. Specific inhibitors of the JNK/pSmad3L pathway might prove useful in this respect. In the case of molecularly targeted therapy for human cancer, pSmad3L and pSmad3C could be assessed as biomarkers to evaluate the likely benefit from specific inhibition of the JNK/pSmad3L pathway.

Menin interacts with activator of S-phase kinase and regulates TGF-beta signaling

Introduction: MEN1 is the tumor suppressor gene responsible for the multiple endocrine neoplasia type 1 (MEN1) syndrome. Menin, the protein product of the MEN1 gene, is thought to function in the regulation of gene transcription and DNA repair pathways. One of key growth regulatory pathways dependent upon menin expression is TGF-beta. In the absence of menin, TGF-beta signaling is significantly reduced. Through yeast two hybrid studies, we identified an important link between menin and ASK, a key protein involved in starting DNA replication.

STROMAL HYPERPLASIA IN MALE BLADDERS UPON LOSS OF TRANSFORMING GROWTH FACTOR-β SIGNALING IN FIBROBLASTS

Rapid bladder growth associated, partial urethral obstruction and embryonic bladder development entail stromal-epithelial interactions involving signaling by the cytokine transforming growth factor-beta (TGF-beta). However, to our knowledge the role of TGF-beta in bladder stromal hyperplasia and hypertrophy is not understood. In an effort to understand the specific role of TGF-beta signaling in bladder stroma a fibroblast specific conditional knockout mouse of the type II TGF-beta receptor gene, Tgfbr2(/spko), was generated using Cre-lox methodology. Bladders from 18, 7 to 8-week-old mice were harvested for histological and immunohistochemical analysis. Bladders from homozygous Tgfbr2(/spko), male mice showed marked hypertrophy in the lamina propria and smooth muscle layers in the absence of visible or functional bladder obstruction by age 8 weeks. However, age matched female mice of the same genotype maintained bladder architecture similar to that in wild-type littermate male and female controls. Immunohistochemistry for the phosphorylated form of Smad2 indicated a general loss in TGF-beta signaling in the lamina propria of bladders of male and female Tgfbr2(/spko), mice, and yet pronounced alpha-smooth muscle actin expression was noted in male Tgfbr2(/spko), bladders, which is a marker for myofibroblasts. A sex disparity was observed in the Tgfbr2(/spko), mouse model lacking TGF-beta signaling in fibroblasts. Deletion of TGF-beta in males leads to a hypertrophied lamina propria and muscularis externa with myofibroblast differentiation and proliferation. Female homozygous Tgfbr2(/spko), bladders appeared the same as those of wild-type male and female controls. This model suggests a role for stromal TGF-beta signaling with estrogens and androgens in bladder fibrosis.

A functional SNP in CILP, encoding cartilage intermediate layer protein, is associated with susceptibility to lumbar disc disease

Lumbar disc disease (LDD) is caused by degeneration of intervertebral discs of the lumbar spine. One of the most common musculoskeletal disorders, LDD has strong genetic determinants. Using a case-control association study, we identified a functional SNP (1184T --> C, resulting in the amino acid substitution I395T) in CILP, which encodes the cartilage intermediate layer protein, that acts as a modulator of LDD susceptibility. CILP was expressed abundantly in intervertebral discs, and its expression increased as disc degeneration progressed. CILP colocalized with TGF-beta1 in clustering chondrocytes and their territorial matrices in intervertebral discs. CILP inhibited TGF-beta1-mediated induction of cartilage matrix genes through direct interaction with TGF-beta1 and inhibition of TGF-beta1 signaling. The susceptibility-associated 1184C allele showed increased binding and inhibition of TGF-beta1. Therefore, we conclude that the extracellular matrix protein CILP regulates TGF-beta signaling and that this regulation has a crucial role in the etiology and pathogenesis of LDD. Our study also adds to the list of connective tissue diseases that are associated with TGF-beta.

TGF‐β Signaling in Colon Cancer Cells

Transforming growth factor-betas (TGF-betas), cytokines expressed in the colon, play important roles as tumor suppressors and tumor promoters during colorectal carcinogenesis. TGF-beta signaling pathway involves activation of Smad2 and Smad3 by the type I receptor and formation of Smad2/3/4 heteromeric complexes that enter the nucleus to regulate transcription. Most human colorectal cancers are resistant to the tumor suppressor effects of TGF-beta, and a subset of human colorectal cancers have mutations in Smad2 and Smad4. The purpose of this study was to determine whether Smads are required for TGF-beta signaling in colon cancer cells. First, we selected a colon cancer cell line (MC-26) that has a functional TGF-beta signaling pathway. We found that MC-26 cells expressed Smad2, Smad3, and Smad4 mRNAs by reverse transeription-polymerase chain reaction and confirmed that the TGF-beta signaling pathway is functional using a transient transfection assay with 3TP-Lux reporter plasmid. TGF-beta also inhibited cell growth and induced apoptosis in MC-26 cells. When MC-26 cells were transiently transfected with dominant-negative carboxyl-terminal truncation mutants of Smad2, Smad3, and Smad4, TGF-beta-induced 3TP-Lux reporter activity was significantly reduced, suggesting that Smad2, Smad3, and Smad4 are attractive novel therapeutic targets for regulating TGF-beta signaling in colorectal cancers. Because MC-26 cells express TGF-beta activated Smads, have a functional TGF-beta signaling pathway, and are sensitive to the growth inhibitory and apoptotic effects of TGF-beta, they can serve as an excellent model to examine TGF-beta signaling in colorectal cancers.

Negative regulation of transforming growth factor-beta (TGF-beta) signaling by WW domain-containing protein 1 (WWP1).

Smad7 negatively regulates transforming growth factor (TGF)-beta superfamily signaling by binding to activated type I receptors, thereby preventing the phosphorylation of receptor-regulated Smads (R-Smads), as well as by recruiting HECT-type E3 ubiquitin ligases to degrade type I receptors through a ubiquitin-dependent mechanism. To elucidate the regulatory mechanisms of TGF-beta signaling, we searched for novel members of proteins that interact with Smad7 using a yeast two-hybrid system. One of the proteins identified was the WW domain-containing protein 1 (WWP1) that is structurally related to Smad ubiquitin regulatory factors (Smurfs), E3 ubiquitin ligases for Smads and TGF-beta superfamily receptors. Using a TGF-beta-responsive reporter in mammalian cells, we found that WWP1 inhibited transcriptional activities induced by TGF-beta. Similar to Smurfs, WWP1 associated with Smad7 and induced its nuclear export, and enhanced binding of Smad7 to TGF-beta type I receptor to cause ubiquitination and degradation of the receptor. Consistent with these results, WWP1 inhibited phosphorylation of Smad2 induced by TGF-beta. WWP1 thus negatively regulates TGF-beta signaling in cooperation with Smad7. However, unlike Smurfs, WWP1 failed to ubiquitinate R-Smads and SnoN. Importantly, WWP1 and Smurfs were expressed in distinct patterns in human tissues and carcinoma cell lines, suggesting unique pathophysiological roles of WWP1 and Smurfs.

Effect of Nma on growth inhibition by TGF-βa in human gastric carcinoma cell lines

Non-metastatic gene A (nma) has a homologue DNA sequence to a gene of bone morphogenetic proteins and activin membrane-bound inhibitor (BAMBI), which negatively regulates TGF-beta signaling. In this study, we analyzed the functional homology between Nma and BAMBI in human gastric carcinoma cell lines. Various levels of nma mRNA expression were detected by the RT-PCR technique in all human gastric carcinoma cell lines. Then, Nma antisense and sense S-oligodeoxynucleotide (ODN) were used to analyze the response of TGF-beta to cell growth and invasion gastric carcinoma cell lines. The cell growth was inhibited by TGF-beta in Nma antisense S-ODN treatment gastric carcinoma cell lines, MKN28, MKN1, MKN74 and TMK1. TGF-beta reduced cell growth and invasive activity of MKN28 treated with Nma antisense S-ODN in a dose and time-dependent manner. Furthermore, lysates of Nma sense or antisense S-ODN treated MKN28 cells were immunoprecipitated with anti-TGFbetaR-I or anti-TGFbetaR-II antibody. The 29 kDa signal considered as Nma appeared in sense S-ODN treated MKN28 cells immunoprecipitated with anti-TGFbetaR-I. These results indicate that Nma negatively regulates TGF-beta signaling, consequently playing an important role as one of the escape mechanisms from TGF-beta-mediated growth control similarly to BAMBI, and induce cell growth and invasion in human gastric carcinoma cell lines.

Syndecan-2 Regulates Transforming Growth Factor-β Signaling

Transforming growth factor-beta (TGF-beta) has multiple functions including increasing extracellular matrix deposition in fibrosis. It functions through a complex family of cell surface receptors that mediate downstream signaling. We report here that a transmembrane heparan sulfate proteoglycan, syndecan-2 (S2), can regulate TGF-beta signaling. S2 protein increased in the renal interstitium in diabetes and regulated TGF-beta-mediated increased matrix deposition in vitro. Transfection of renal papillary fibroblasts with S2 or a S2 construct that has a truncated cytoplasmic domain (S2DeltaS) promoted TGF-beta binding and S2 core protein ectodomain directly bound TGF-beta. Transfection with S2 increased the amounts of type I and type II TGF-beta receptors (TbetaRI and TbetaRII), whereas S2DeltaS was much less effective. In contrast, S2DeltaS dramatically increased the level of type III TGF-beta receptor (TbetaRIII), betaglycan, whereas S2 resulted in a decrease. Syndecan-2 specifically co-immunoprecipitated with betaglycan but not with TbetaRI or TbetaRII. This is a novel mechanism of control of TGF-beta action that may be important in fibrosis.

Sumoylation of Smad4, the Common Smad Mediator of Transforming Growth Factor-β Family Signaling

Transforming growth factor-beta (TGF-beta) and TGF-beta-related factors regulate cell growth, differentiation, and apoptosis, and play key roles in normal development and tumorigenesis. TGF-beta family-induced changes in gene expression are mediated by serine/threonine kinase receptors at the cell surface and Smads as intracellular effectors. Receptor-activated Smads combine with a common Smad4 to translocate into the nucleus where they cooperate with other transcription factors to activate or repress transcription. The activities of the receptor-activated Smads are controlled by post-translational modifications such as phosphorylation and ubiquitylation. Here we show that Smad4 is modified by sumoylation. Sumoylation of Smad4 was enhanced by the conjugating enzyme Ubc9 and members of the PIAS family of SUMO ligases. A major sumoylation site in Smad4 was localized to Lys-159 in its linker segment with an additional site at Lys-113 in the MH-1 domain. Increased sumoylation in the presence of the PIASy E3 ligase correlated with targeting of Smad4 to subnuclear speckles that contain SUMO-1 and PIASy. Replacement of lysines 159 and 113 by arginines or increased sumoylation enhanced the stability of Smad4, and transcription in mammalian cells and Xenopus embryos. These observations suggest a role for Smad4 sumoylation in the regulation of TGF-beta signaling through Smads.

Endoglin is expressed on human chondrocytes and forms a heteromeric complex with betaglycan in a ligand and type II TGFbeta receptor independent manner.

Previous work has implicated transforming growth factor beta (TGFbeta) as an essential mediator of cartilage repair and TGFbeta signaling as a requirement for the maintenance of articular cartilage in vivo. However, the mechanisms regulating TGFbeta action in chondrocytes are poorly understood. Endoglin, an accessory receptor of the TGFbeta receptor superfamily, is highly expressed on endothelial cells and has been shown to potently modulate TGFbeta responses. It is not known whether chondrocytes express endoglin or whether it modulates TGFbeta signaling in these cells. In this study, we show that endoglin is expressed on human chondrocytes at levels comparable with endothelial cells and that it forms higher order complexes with the types I and II TGFbeta receptors. More importantly, we show that endoglin forms a heteromeric complex with betaglycan on these cells at endogenous receptor concentrations and ratios. Endoglin complexes with betaglycan in a ligand-independent and -dependent manner as indicated by co-immunoprecipitation in the absence of TGFbeta and after affinity labeling with radiolabeled TGFbeta, respectively. Also, the endoglin-betaglycan association can occur independently of the type II TGFbeta receptor. These findings, taken together with the available evidence that endoglin and betaglycan are potent modulators of TGFbeta signal transduction, imply that the complex formation between endoglin and betaglycan may be of critical significance in the regulation of TGFbeta signaling in chondrocytes.

Human T-cell Lymphotropic Virus Type 1 Tax Inhibits Transforming Growth Factor-β Signaling by Blocking the Association of Smad Proteins with Smad-binding Element

The human T-cell lymphotropic virus type 1 (HTLV-1) oncoprotein Tax is implicated in various clinical manifestations associated with infection by HTLV-1, including an aggressive and fatal T-cell malignancy. Because many human HTLV-1-infected T-cell lines are resistant to the growth inhibitory activity of transforming growth factor beta (TGF-beta), we examined the possibility that the HTVL-1-Tax oncoprotein regulates TGF-beta signaling. We show that Tax significantly decreases transcriptional activity and growth inhibition in response to TGF-beta. Tax inhibits TGF-beta-induced plasminogen activator inhibitor-1 expression and Smad2 phosphorylation. Competitive interaction studies show that Tax inhibits TGF-beta signaling, in part, by disrupting the interaction of the Smads with the transcriptional co-activator p300. Tax directly interacts with Smad2, Smad3, and Smad4; the Smad MH2 domain binds to Tax. Furthermore, Tax inhibits Smad3.Smad4 complex formation and its DNA binding. These results suggest that suppression of Smad-mediated signaling by Tax may contribute to HTLV-1-associated leukemogenesis.

Functional proteomics of transforming growth factor-beta1-stimulated Mv1Lu epithelial cells: Rad51 as a target of TGFbeta1-dependent regulation of DNA repair.

Transforming growth factor-beta (TGFbeta) conveys regulatory signals through multiple intracellular pathways, subsequently affecting various cellular functions. To identify new targets for TGFbeta, we studied the changes in the proteome of Mv1Lu lung epithelial cells in response to TGFbeta1 treatment. Thirty-eight non-abundant protein spots, affected by TGFbeta1, were selected, and proteins were identified by peptide mass-fingerprinting (PMF). Among them, proteins involved in regulation of immune response, apoptosis, regulation of TGFbeta signalling, metabolism and DNA repair were identified. Twenty-eight of the 38 proteins are new targets for TGFbeta1, thus suggesting novel ways of integration of TGFbeta signalling in intracellular regulatory processes. We show that TGFbeta1-dependent decrease in expression of one of the new targets, Rad51, correlates with a decrease in DNA repair efficiency. This was evaluated by formation of nuclear Rad51-containing DNA repair complexes in response to DNA damage, by single cell gel electrophoresis and by cell survival assay. The TGFbeta1-dependent inhibition of DNA repair was reversed by ectopic overexpression of Rad51. Therefore, TGFbeta can promote DNA instability through down-regulation of Rad51 and inhibition of DNA repair.

Phosphorylation of Smad7 at Ser-249 Does Not Interfere with Its Inhibitory Role in Transforming Growth Factor-β-dependent Signaling but Affects Smad7-dependent Transcriptional Activation

Smad proteins are major components in the intracellular signaling pathway of transforming growth factor-beta (TGF-beta), and phosphorylation is an important mechanism in regulation of their functions. Smad7 was identified as a potent inhibitor of TGF-beta-dependent signaling. We have identified serine 249 in Smad7 as a major phosphorylation site, the phosphorylation of which was not affected by TGF-beta1. Abrogation of the phosphorylation by substitution of Ser-249 with alanine or aspartic acid residues did not affect the ability of Smad7 to inhibit TGF-beta1 and BMP7 signaling. No differences were found in the stability or in the intracellular distribution of Smad7 mutants compared with the wild-type molecule. However, Smad7 fused to the DNA-binding domain of GAL4 induced transcription from a reporter with mutated TATA minimal promoter in a Ser-249-dependent manner. Moreover, a reporter with the SV40 minimal promoter was inhibited by GAL4-Smad7, and this effect was also dependent on Ser-249 phosphorylation. The amplitude of effects on transcriptional regulation was dependent on cell type. Our results suggest that phosphorylation of Smad7, unlike phosphorylation of the receptor-regulated Smads, does not regulate TGF-beta signaling but rather affects TGF-beta-independent effects of Smad7 on transcriptional regulation.

Functional Roles for the Cytoplasmic Domain of the Type III Transforming Growth Factor β Receptor in Regulating Transforming Growth Factor β Signaling

Transforming growth factor beta (TGF-beta) signals through three high affinity cell surface receptors, TGF-beta type I, type II, and type III receptors. The type III receptor, also known as betaglycan, binds to the type II receptor and is thought to act solely by "presenting" the TGF-beta ligand to the type II receptor. The short cytoplasmic domain of the type III receptor is thought to have no role in TGF-beta signaling because deletion of this domain has no effect on association with the type II receptor, or with the presentation role of the type III receptor. Here we demonstrate that the cytoplasmic domains of the type III and type II receptors interact specifically in a manner dependent on the kinase activity of the type II receptor and the ability of the type II receptor to autophosphorylate. This interaction results in the phosphorylation of the cytoplasmic domain of the type III receptor by the type II receptor. The type III receptor with the cytoplasmic domain deleted is able to bind TGF-beta, to bind the type II receptor, and to enhance TGF-beta binding to the type II receptor but is unable to enhance TGF-beta2 signaling, determining that the cytoplasmic domain is essential for some functions of the type III receptor. The type III receptor functions by selectively binding the autophosphorylated type II receptor via its cytoplasmic domain, thus promoting the preferential formation of a complex between the autophosphorylated type II receptor and the type I receptor and then dissociating from this active signaling complex. These studies, for the first time, elucidate important functional roles of the cytoplasmic domain of the type III receptor and demonstrate that these roles are essential for regulating TGF-beta signaling.

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

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 was recently identified to antagonize 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 reduced epithelial lung branching morphogenesis in control virus-infected lung culture, TGF-beta-induced branching inhibition was abolished after epithelial transfer of the Smad7 gene into lungs in culture. Smad7 also prevented TGF-beta-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-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 were not observed in cultured embryonic lungs with Smad6 adenoviral gene transfer, suggesting that Smad7 and Smad6 differentially regulate TGF-beta signaling in developing lungs. Our 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.