Inhibition of Transforming Growth Factor β-enhanced Serum Response Factor-dependent Transcription by SMAD7 (original) (raw)
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The role of the TGF-SMAD signalling pathway in the etiopathogenesis of severe asthma
Pneumonologia i alergologia polska, 2016
Asthma is a chronic inflammatory heterogeneous disease of the lower respiratory tract characterised by the occurrence of bronchial hyper-responsiveness and paroxysmal, changeable bronchial obstruction. Transforming growth factor-beta (TGF-b) is one of the cytokines involved in mediating airway inflammation and remodelling. The level of TGF-b1 gene expression correlates with severity of symptoms. Alterations in the main SMAD signal transmission, overexpression of TGF-b genes and changes in the transcriptome cause excessive secretion of TGF-b and its increased expression in target cells, which clinically induces a moderate-severe or severe course of asthma as well as an earlier and faster disease progression. Knowledge of these processes allows clinicians to assess immune responses in patients, which affects adequate disease control and prevention of remodelling.
Journal of Allergy and Clinical Immunology, 2002
Background: Smad7 is an intracellular antagonist of transforming growth factor β (TGF-β) signaling, which could determine the intensity or duration of the TGF-β signal. Because TGF-β has been implicated in the development of airway remodeling in asthma on the basis of its strong capacity to induce extracellular matrix production, it is possible that Smad7 also plays some roles in the regulation of the process. Objective: We sought to determine the relationships between Smad7 expression in bronchial biopsy samples from asthmatic subjects and clinicopathologic features. Methods: Bronchial biopsy specimens were obtained from 40 asthmatic subjects and 6 healthy control subjects. Expression levels of Smad7 on a histologic section were estimated by immunohistochemical staining. In addition, the roles of Smad7 in TGF-β-mediated transcriptional responses were studied by in vitro studies. Results: Smad7 immunoreactivity was detected mainly in bronchial epithelial cells in control and asthmatic subjects. Interestingly, asthmatic subjects exhibited less Smad7 immunoreactivity in bronchial epithelial cells than normal subjects. Expression levels of Smad7 in bronchial epithelial cells were inversely correlated with basement membrane thickness and airway hyperresponsiveness in asthmatic subjects. In addition, abrogation of endogenous Smad7 expression through use of an antisense oligonucleotide enhanced transcriptional responses to TGF-β, whereas overexpression of Smad7 inhibited TGF-β-induced plasminogen activator inhibitor 1production in a human bronchial epithelial cell line, BEAS2B cells. Conclusion: These findings suggest that Smad7 is a key molecule that defines the susceptibility of bronchial epithelial cells to TGF-β action and that regulation of Smad7 expression in bronchial epithelial cells might be related to the development of airway remodeling and airway hyperresponsiveness in asthma. (J Allergy Clin Immunol 2002;110:873-8.)
Activation of TGF-β/Smad2 signaling is associated with airway remodeling in asthma
Journal of Allergy and Clinical Immunology, 2002
Background: Transforming growth factor β (TGF-β) has been suggested to play an important role in the development of airway remodeling in asthma; this suggestion is based on evidence that expression levels of TGF-β are correlated with unique parameters of airway remodeling, such as thickness of basement membrane. However, the relevant studies were inconclusive because they were unable to demonstrate active signaling mediated by the cytokine in the airways of asthmatic individuals. Objective: We sought to determine whether TGF-β signaling was active in the airways of asthmatic subjects and, if so, whether it was correlated with clinicopathologic features associated with the development of airway remodeling in asthma. Methods: We examined the phosphorylation status of Smad2 in bronchial biopsy samples obtained from 40 asthmatic subjects as a marker of active TGF-β signaling, and we studied its correlation with basement membrane thickness. Results: Expression levels of phosphorylated Smad2 in bronchial biopsy specimens from asthmatic subjects were higher than those in specimens from normal subjects, and they were correlated with basement membrane thickness in asthma. Conclusion: The findings provide evidence that TGF-β signaling was active in asthmatic airways and that the activity was associated with the development of airway remodeling in asthma. (J Allergy Clin Immunol 2002;110:249-54.)
Journal of Biological Chemistry, 2000
Smad7 is an inducible intracellular inhibitor of transforming growth factor- (TGF-) signaling that is regulated by diverse stimuli including members of the TGF- superfamily. To define the molecular mechanisms of negative control of TGF- signaling, we have isolated the human SMAD7 gene and characterized its promoter region. A ؊303 to ؉672 SMAD7 region contained a palindromic GTCTAGAC Smad binding element (SBE) between nucleotides ؊179 and ؊172 that was necessary for the induction of a Smad7 promoter luciferase reporter gene by TGF-. Electrophoretic mobility shift assays using oligonucleotide probes demonstrated that TGF- rapidly induced the binding of an endogenous SBEbinding complex (SBC) containing Smad2, Smad3, and Smad4. Transfection assays in mouse embryonic fibroblasts (MEFs), with targeted deletions of either Smad2 or Smad3, and the Smad4-deficient cell line MD-MBA-468 revealed that both Smad3 and Smad4, but not Smad2, were absolutely required for induction of the Smad7 promoter reporter gene by TGF-. Furthermore, the TGF--inducible SBE-binding complex was diminished in Smad2-deficient MEFs when compared with wild type MEFs and not detectable in Smad3-deficient MEFs and MD-MBA-468 cells. Taken together, our data demonstrate that TGF- induces transcription of the human SMAD7 gene through activation of Smad3 and Smad4 transcription factor binding to its proximal promoter.
Smad7 Differentially Regulates Transforming Growth Factor β-mediated Signaling Pathways
Journal of Biological Chemistry, 1999
Smad7 has been identified as a negative regulator of transforming growth factor  (TGF-) signaling by interfering with the phosphorylation of other Smad proteins by TGF- receptor type I (TRI). We established a mink lung epithelial (Mv1Lu) cell line where ectopic expression of Smad7 is tightly controlled by doxycycline using an improved Tet-on system. Once induced by doxycycline, the recombinant Smad7 was localized predominantly in the perinuclear region and in the cytoplasm. However, the type of culture surface alters the subcellular localization of Smad7: on plastic or on fibronectin-coated glass, Smad7 was localized in the cytoplasm; but when the cells were cultured on glass, nuclear localization was observed. TGF- stimulation did not alter substantially the cellular distribution of Smad7. Importantly, the expression of recombinant Smad7 differentially inhibited TGF- signaling pathways. Consistent with previous studies, Smad7 inhibited TGF--stimulated induction of type 1 plasminogen activator inhibitor as measured by p3TP-Lux reporter. However, expression of Smad7 had little effect on TGF-induced growth inhibition.
Smad7 Differentially Regulates Transforming Growth Factor beta -mediated Signaling Pathways
Journal of Biological Chemistry, 1999
Smad7 has been identified as a negative regulator of transforming growth factor  (TGF-) signaling by interfering with the phosphorylation of other Smad proteins by TGF- receptor type I (TRI). We established a mink lung epithelial (Mv1Lu) cell line where ectopic expression of Smad7 is tightly controlled by doxycycline using an improved Tet-on system. Once induced by doxycycline, the recombinant Smad7 was localized predominantly in the perinuclear region and in the cytoplasm. However, the type of culture surface alters the subcellular localization of Smad7: on plastic or on fibronectin-coated glass, Smad7 was localized in the cytoplasm; but when the cells were cultured on glass, nuclear localization was observed. TGF- stimulation did not alter substantially the cellular distribution of Smad7. Importantly, the expression of recombinant Smad7 differentially inhibited TGF- signaling pathways. Consistent with previous studies, Smad7 inhibited TGF--stimulated induction of type 1 plasminogen activator inhibitor as measured by p3TP-Lux reporter. However, expression of Smad7 had little effect on TGF-induced growth inhibition.
Genomic locus and promoter region of rat Smad7, an important antagonist of TGFβ signaling
Mammalian Genome, 2000
SMAD proteins are essential components of the intracellular signaling pathways utilized by members of the transforming growth factor  (TGF) superfamily of growth factors. Certain SMAD proteins (Smad1, 2, 3, and 5) can act as regulated transcriptional activators. This process involves phosphorylation of these proteins by activated TGF receptors. Recently, Smad6 and Smad7 were identified; they antagonize TGF signaling by preventing the activation of signal-transducing SMAD complexes. TGF rapidly induces the expression of Smad7 mRNA, suggesting participation of Smad7 in a negative feedback loop to control TGF responses. Similarly, epidermal growth factor (EGF) and interferon ␥ (IFN␥) have been reported to induce Smad7 expression. In a rat model system of liver fibrosis, TGF inducibility of Smad7 is abrogated during transformation of hepatic stellate cells (HSC), indicating an important switch in transcriptional regulation of the gene. With the detailed characterization of the rat Smad7 genomic organization including the promoter region, we present the first identified Smad7 gene so far. 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 were 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 were identified, which could be the targets of TGF, IFN␥, and EGFdependent Smad7 transcription initiation.
The Journal of Immunology, 2005
Airway inflammation and remodeling are important pathophysiologic features of chronic asthma. Previously, we have developed a mouse model of prolonged allergen challenge which exhibits many characteristics of chronic asthma such as goblet cell hyperplasia and subepithelial collagen deposition, in association with an increase in lung expression of the profibrotic mediator, TGF-. The aim of this study was to determine the effects of blockade of TGF- on the development of airway inflammation and remodeling using our murine model of prolonged allergen challenge. Importantly anti-TGF- Ab was administered therapeutically, with dosing starting after the onset of established eosinophilic airway inflammation. Therapeutic treatment of mice with anti-TGF- Ab significantly reduced peribronchiolar extracellular matrix deposition, airway smooth muscle cell proliferation, and mucus production in the lung without affecting established airway inflammation and Th2 cytokine production. Thus, our data suggest that it might be possible to uncouple airway inflammation and remodeling during prolonged allergen challenge. In addition, anti-TGF- Ab treatment was shown to regulate active TGF- signaling in situ with a reduction in the expression of phospho-Smad 2 and the concomitant up-regulation of Smad 7 in lung sections. Therefore, this is the first report to suggest that anti-TGF- Ab treatment prevents the progression of airway remodeling following allergen challenge even when given in a therapeutic mode. Moreover, the molecular mechanism behind this effect may involve regulation of active TGF- signaling. The Journal of Immunology, 2005, 174: 5774 -5780.
Journal of Biological Chemistry, 2001
Smad proteins are major components in the intracellular signaling pathway of transforming growth factor- (TGF-), and phosphorylation is an important mechanism in regulation of their functions. Smad7 was identified as a potent inhibitor of TGF--dependent signaling. We have identified serine 249 in Smad7 as a major phosphorylation site, the phosphorylation of which was not affected by TGF-1. 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-1 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- signaling but rather affects TGF--independent effects of Smad7 on transcriptional regulation.
Protocadherin-1 binds to SMAD3 and suppresses TGFβ1-induced gene transcription
American Journal of Physiology - Lung Cellular and Molecular Physiology, 2015
Genetic studies have identified Protocadherin-1 ( PCDH1) and Mothers against decapentaplegic homolog-3 ( SMAD3) as susceptibility genes for asthma. PCDH1 is expressed in bronchial epithelial cells and has been found to interact with SMAD3 in yeast two-hybrid (Y2H) overexpression assays. Here, we test whether PCDH1 and SMAD3 interact at endogenous protein levels in bronchial epithelial cells and evaluate the consequences thereof for transforming growth factor-β1 (TGF-β1)-induced gene transcription. We performed Y2H screens and coimmunoprecipitation (co-IP) experiments of PCDH1 and SMAD3 in HEK293T and 16HBE14o− (16HBE) cell lines. Activity of a SMAD3-driven luciferase reporter gene in response to TGF-β1 was measured in BEAS-2B cells transfected with PCDH1 and in 16HBE cells transfected with PCDH1-small-interfering RNA (siRNA). TGF-β1-induced gene expression was quantified in BEAS-2B clones overexpressing PCDH1 and in human primary bronchial epithelial cells (PBECs) transfected with P...