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1.  Early Growth Response Gene 1–mediated Apoptosis Is Essential for Transforming Growth Factor β1–induced Pulmonary Fibrosis 
Fibrosis and apoptosis are juxtaposed in pulmonary disorders such as asthma and the interstitial diseases, and transforming growth factor (TGF)-β1 has been implicated in the pathogenesis of these responses. However, the in vivo effector functions of TGF-β1 in the lung and its roles in the pathogenesis of these responses are not completely understood. In addition, the relationships between apoptosis and other TGF-β1–induced responses have not been defined. To address these issues, we targeted bioactive TGF-β1 to the murine lung using a novel externally regulatable, triple transgenic system. TGF-β1 produced a transient wave of epithelial apoptosis that was followed by mononuclear-rich inflammation, tissue fibrosis, myofibroblast and myocyte hyperplasia, and septal rupture with honeycombing. Studies of these mice highlighted the reversibility of this fibrotic response. They also demonstrated that a null mutation of early growth response gene (Egr)-1 or caspase inhibition blocked TGF-β1–induced apoptosis. Interestingly, both interventions markedly ameliorated TGF-β1–induced fibrosis and alveolar remodeling. These studies illustrate the complex effects of TGF-β1 in vivo and define the critical role of Egr-1 in the TGF-β1 phenotype. They also demonstrate that Egr-1–mediated apoptosis is a prerequisite for TGF-β1–induced fibrosis and remodeling.
doi:10.1084/jem.20040104
PMCID: PMC2211975  PMID: 15289506
asthma; pulmonary fibrosis; fibrosis reversibility; airway remodeling
2.  Transgenic Modeling of Transforming Growth Factor-β1 
Inflammation and tissue remodeling with pathologic fibrosis are common consequences of Th2 responses in the lung and other organs. Interleukin (IL)-13 and transforming growth factor-β1 (TGF-β1) are frequently coexpressed in these responses and are believed to play important roles in the pathogenesis of Th2-induced pathologies. To shed light on the mechanisms of these responses, overexpression transgenic approaches were used to selectively target each of these cytokines to the murine lung. IL-13 proved to be a potent stimulator of eosinophilic inflammation, mucus metaplasia, tissue fibrosis, and alveolar remodeling. CC chemokines, specific chemokine receptors (CCR2, CCR1), adenosine metabolism, vascular endothelial growth factor, and IL-11 contributed to the genesis of these responses. IL-13 also induced tissue fibrosis, at least in part, via its ability to induce and activate TGF-β1. In the TGF-β1 transgenic mouse, epithelial apoptosis preceded the onset of tissue fibrosis and alveolar remodeling. In addition, chemical (Z-VAD-fmk) and genetic (null mutations of early growth response gene 1) interventions blocked apoptosis and ameliorated TGF-β1–induced fibrosis and alveolar restructuring. These studies define an IL-13–TGF-β1 pathway of tissue remodeling that regulates inflammation, mucus metaplasia, apoptosis, vascular responses, and fibrosis in the lung. They also highlight the intimate relationship between apoptosis and fibrosis induced by TGF-β1. By defining the complexities of this pathway, these studies highlight sites at which therapies can be directed to control these important responses.
doi:10.1513/pats.200602-017AW
PMCID: PMC2658706  PMID: 16799085
asthma; fibrosis; interleukin-13; transforming growth factor-β; 1; transgenic
3.  Regulation of the effects of TGF-β1 by activation of latent TGF-β1 and differential expression of TGF-β receptors (TβR-I and TβR-II) in idiopathic pulmonary fibrosis 
Thorax  2001;56(12):907-915.
BACKGROUND—Idiopathic pulmonary fibrosis (IPF) is characterised by subpleural fibrosis that progresses to involve all areas of the lung. The expression of transforming growth factor-β1 (TGF-β1), a potent regulator of connective tissue synthesis, is increased in lung sections of patients with IPF. TGF-β1 is generally released in a biologically latent form (L-TGF-β1). Before being biologically active, TGF-β must be converted to its active form and interact with both TGF-β receptors type I and II (TβR-I and TβR-II). TGF-β latency binding protein 1 (LTBP-1), which facilitates the release and activation of L-TGF-β1, is also important in the biology of TGF-β1.
METHODS—Open lung biopsy samples from patients with IPF and normal controls were examined to localise TβR-I, TβR-II, and LTBP-1. Alveolar macrophages (AM) and bronchoalveolar lavage (BAL) fluid were examined using the CCL-64 bioassay to determine if TGF-β is present in its active form in the lungs of patients with IPF.
RESULTS—Immunoreactive L-TGF-β1 was present in all lung cells of patients with IPF except for fibroblasts in the subepithelial regions of honeycomb cysts. LTBP-1 was detected primarily in AM and epithelial cells lining honeycomb cysts in areas of advanced IPF. In normal lungs LTBP-1 immunoreactivity was observed in a few AM. AM from the upper and lower lobes of patients with IPF secreted 1.6 (0.6) fmol and 4.1 (1.9) fmol active TGF-β, respectively, while AM from the lower lobes of control patients secreted no active TGF-β (p⩽0.01 for TGF-β in the conditioned media from AM obtained from the lower lobes of IPF patients v normal controls). The difference in percentage active TGF-β secreted by AM from the lower lobes of patients with IPF and the lower lobes of control patients was significant (p⩽0.01), but the difference between the total TGF-β secreted from these lobes was not significant. The difference in active TGF-β in conditioned media of AM from the upper and lower lobes of patients with IPF was also not statistically significant. BAL fluid from the upper and lower lobes of patients with IPF contained 0.7 (0.2) fmol and 2.9 (1.2) fmol active TGF-β, respectively (p⩽0.03). The percentage of active TGF-β in the upper and lower lobes was 17.6 (1.0)% and 78.4 (1.6)%, respectively (p⩽0.03). In contrast, BAL fluid from control patients contained small amounts of L-TGF-β. Using immunostaining, both TβR-I and TβR-II were present on all cells of normal lungs but TβR-I was markedly reduced in most cells in areas of honeycomb cysts except for interstitial myofibroblasts in lungs of patients with IPF. TGF-β1 inhibits epithelial cell proliferation and a lack of TβR-I expression by epithelial cells lining honeycomb cysts would facilitate repair of the alveoli by epithelial cell proliferation. However, the presence of both TβRs on fibroblasts is likely to result in a response to TGF-β1 for synthesis of connective tissue proteins. Our findings show that biologically active TGF-β1 is only present in the lungs of patients with IPF. In addition, the effects of TGF-β1 on cells may be further regulated by the expression of TβRs.
CONCLUSION—Activation of L-TGF-β1 and the differential expression of TβRs may be important in the pathogenesis of remodelling and fibrosis in IPF.


doi:10.1136/thorax.56.12.907
PMCID: PMC1745982  PMID: 11713352
4.  Syndecan-2 Exerts Antifibrotic Effects by Promoting Caveolin-1–mediated Transforming Growth Factor-β Receptor I Internalization and Inhibiting Transforming Growth Factor-β1 Signaling 
Rationale: Alveolar transforming growth factor (TGF)-β1 signaling and expression of TGF-β1 target genes are increased in patients with idiopathic pulmonary fibrosis (IPF) and in animal models of pulmonary fibrosis. Internalization and degradation of TGF-β receptor TβRI inhibits TGF-β signaling and could attenuate development of experimental lung fibrosis.
Objectives: To demonstrate that after experimental lung injury, human syndecan-2 confers antifibrotic effects by inhibiting TGF-β1 signaling in alveolar epithelial cells.
Methods: Microarray assays were performed to identify genes differentially expressed in alveolar macrophages of patients with IPF versus control subjects. Transgenic mice that constitutively overexpress human syndecan-2 in macrophages were developed to test the antifibrotic properties of syndecan-2. In vitro assays were performed to determine syndecan-2–dependent changes in epithelial cell TGF-β1 signaling, TGF-β1, and TβRI internalization and apoptosis. Wild-type mice were treated with recombinant human syndecan-2 during the fibrotic phase of bleomycin-induced lung injury.
Measurements and Main Results: We observed significant increases in alveolar macrophage syndecan-2 levels in patients with IPF. Macrophage-specific overexpression of human syndecan-2 in transgenic mice conferred antifibrotic effects after lung injury by inhibiting TGF-β1 signaling and downstream expression of TGF-β1 target genes, reducing extracellular matrix production and alveolar epithelial cell apoptosis. In vitro, syndecan-2 promoted caveolin-1–dependent internalization of TGF-β1 and TβRI in alveolar epithelial cells, which inhibited TGF-β1 signaling and epithelial cell apoptosis. Therapeutic administration of human syndecan-2 abrogated lung fibrosis in mice.
Conclusions: Alveolar macrophage syndecan-2 exerts antifibrotic effects by promoting caveolin-1–dependent TGF-β1 and TβRI internalization and inhibiting TGF-β1 signaling in alveolar epithelial cells. Hence, molecules that facilitate TβRI degradation via endocytosis represent potential therapies for pulmonary fibrosis.
doi:10.1164/rccm.201303-0434OC
PMCID: PMC3826270  PMID: 23924348
idiopathic pulmonary fibrosis; TGF-β1 signaling; syndecan-2; alveolar macrophage
5.  Epithelial-mesenchymal transition in primary human bronchial epithelial cells is Smad-dependent and enhanced by fibronectin and TNF-α 
Background
Defective epithelial repair, excess fibroblasts and myofibroblasts, collagen overproduction and fibrosis occur in a number of respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD) and pulmonary fibrosis. Pathological conversion of epithelial cells into fibroblasts (epithelial-mesenchymal transition, EMT) has been proposed as a mechanism for the increased fibroblast numbers and has been demonstrated to occur in lung alveolar epithelial cells. Whether other airway cell types also have the capability to undergo EMT has been less explored so far. A better understanding of the full extent of EMT in airways, and the underlying mechanisms, can provide important insights into airway disease pathology and enable the development of new therapies. The main aim of this study was to test whether primary human bronchial epithelial cells are able to undergo EMT in vitro and to investigate the effect of various profibrotic factors in the process.
Results
Our data demonstrate that primary human bronchial epithelial cells (HBECs) are able to undergo EMT in response to transforming growth factor-beta 1 (TGF-β1), as revealed by typical morphological alterations and EMT marker progression at the RNA level by real-time quantitative polymerase chain reaction and, at the protein level, by western blot. By using pharmacological inhibitors we show that this is a Smad-dependent mechanism and is independent of extracellular signal-related kinase pathway activation. Additional cytokines and growth factors such as tumour necrosis factor-alpha (TNF-α), interleukin-1 beta (IL1β) and connective tissue growth factor (CTGF) were also tested, alone or in combination with TGF-β1. TNF-α markedly enhances the effect of TGF-β1 on EMT, whereas IL1β shows only a very weak effect and CTGF has no significant effect. We have also found that cell-matrix contact, in particular to fibronectin, an ECM component upregulated in fibrotic lesions, potentiates EMT in both human alveolar epithelial cells and HBECs. Furthermore, we also show that the collagen discoidin domain receptor 1 (DDR1), generally expressed in epithelial cells, is downregulated during the EMT of bronchial epithelium whereas DDR2 is unaffected. Our results also suggest that bone morphogenetic protein-4 is likely to have a context dependent effect during the EMT of HBECs, being able to induce the expression of EMT markers and, at the same time, to inhibit TGF-β induced epithelial transdifferentiation.
Conclusions
The results presented in this study provide additional insights into EMT, a potentially very important mechanism in fibrogenesis. We show that, in addition to alveolar epithelial type II cells, primary HBECs are also able to undergo EMT in vitro upon TGF-β1 stimulation via a primarily Smad 2/3 dependent mechanism. The effect of TGF-β1 is potentiated on fibronectin matrix and in the presence of TNF-α, representing a millieu reminiscent of fibrotic lesions. Our results can contribute to a better understanding of lung fibrosis and to the development of new therapeutic approaches.
doi:10.1186/1755-1536-3-2
PMCID: PMC2821296  PMID: 20051102
6.  A potential role of the JNK pathway in hyperoxia-induced cell death, myofibroblast transdifferentiation and TGF-β1-mediated injury in the developing murine lung 
BMC Cell Biology  2011;12:54.
Background
Transforming growth factor-beta 1 (TGF-β1) has been implicated in hyperoxia-induced cell death and impaired alveolarization in the developing lung. In addition, the c-JunNH2-terminal kinase (JNK) pathway has been shown to have a role for TGF-β1-mediated effects. We hypothesized that the JNK pathway is an important regulator of hyperoxia-induced pulmonary responses in the developing murine lung.
Results
We used cultured human lung epithelial cells, fetal rat lung fibroblasts and a neonatal TGF-β1 transgenic mouse model. We demonstrate that hyperoxia inhibits cell proliferation, activates cell death mediators and causes cell death, and promotes myofibroblast transdifferentiation, in a dose-dependent manner. Except for fibroblast proliferation, the effects were mediated via the JNK pathway. In addition, since we observed increased expression of TGF-β1 by epithelial cells on exposure to hyperoxia, we used a TGF-β1 transgenic mouse model to determine the role of JNK activation in TGF-β1 induced effects on lung development and on exposure to hyperoxia. We noted that, in this model, inhibition of JNK signaling significantly improved the spontaneously impaired alveolarization in room air and decreased mortality on exposure to hyperoxia.
Conclusions
When viewed in combination, these studies demonstrate that hyperoxia-induced cell death, myofibroblast transdifferentiation, TGF-β1- and hyperoxia-mediated pulmonary responses are mediated, at least in part, via signaling through the JNK pathway.
doi:10.1186/1471-2121-12-54
PMCID: PMC3266206  PMID: 22172122
7.  Semaphorin 7A plays a critical role in TGF-β1–induced pulmonary fibrosis 
The Journal of Experimental Medicine  2007;204(5):1083-1093.
Semaphorin (SEMA) 7A regulates neuronal and immune function. In these studies, we tested the hypothesis that SEMA 7A is also a critical regulator of tissue remodeling. These studies demonstrate that SEMA 7A and its receptors, plexin C1 and β1 integrins, are stimulated by transforming growth factor (TGF)-β1 in the murine lung. They also demonstrate that SEMA 7A plays a critical role in TGF-β1–induced fibrosis, myofibroblast hyperplasia, alveolar remodeling, and apoptosis. TGF-β1 stimulated SEMA 7A via a largely Smad 3–independent mechanism and stimulated SEMA 7A receptors, matrix proteins, CCN proteins, fibroblast growth factor 2, interleukin 13 receptor components, proteases, antiprotease, and apoptosis regulators via Smad 2/3–independent and SEMA 7A–dependent mechanisms. SEMA 7A also played an important role in the pathogenesis of bleomycin-induced pulmonary fibrosis. TGF-β1 and bleomycin also activated phosphatidylinositol 3-kinase (PI3K) and protein kinase B (PKB)/AKT via SEMA 7A–dependent mechanisms, and PKB/AKT inhibition diminished TGF-β1–induced fibrosis. These observations demonstrate that SEMA 7A and its receptors are induced by TGF-β1 and that SEMA 7A plays a central role in a PI3K/PKB/AKT-dependent pathway that contributes to TGF-β1–induced fibrosis and remodeling. They also demonstrate that the effects of SEMA 7A are not specific for transgenic TGF-β1, highlighting the importance of these findings for other fibrotic stimuli.
doi:10.1084/jem.20061273
PMCID: PMC2118575  PMID: 17485510
8.  Paclitaxel interrupts TGF-β1 signaling between gallbladder epithelial cells and myofibroblasts 
The Journal of surgical research  2007;141(2):183-191.
Background
The cellular and molecular mechanisms of fibrogenesis in the extrahepatic biliary epithelium are not known. TGF-β1 is a cytokine implicated in signaling pathways that mediate collagen formation. An observation that paclitaxel (PT), applied topically into the rat common bile duct, inhibited stricture formation led us to hypothesize that PT’s effects might be due to interruption of TGF-β1 signaling between biliary epithelial cells and subepithelial myofibroblasts.
Materials and methods
We tested this hypothesis using an in vitro cell culture model in which murine gallbladder epithelial cells (GBEC) are cultured separately or co-cultured with human gallbladder myofibroblasts (GBMF).
Results
Exposure to E. coli lipopolysaccharide (LPS) enhanced TGF-β1 mRNA expression, and stimulated TGF-β1 protein secretion into both apical and basolateral compartments in GBEC. This effect was more prominent with basolateral secretion, and was also more pronounced in the co-culture system. In GBMF, collagen I mRNA expression and protein secretion were stimulated by treatment with LPS or TGF-β1. GBMF also expressed TGF-β1 mRNA, whose levels were enhanced by exposure to either LPS or exogenous TGF-β1. PT inhibited LPS-induced TGF-β1 mRNA expression and protein secretion in GBEC in both culture systems. TNF-α mRNA expression and protein secretion were not affected by PT in GBEC, demonstrating that the effects were specific for TGF-β1. PT also inhibited LPS- and TGF-β1-induced collagen I mRNA expression and protein secretion in GBMF.
Conclusions
These findings support a model in which GBEC communicate with subepithelial GBMF via TGF-β1, leading to collagen deposition and fibrosis; and in which GBMF possess autocrine mechanisms involving TGF-β1 that could regulate collagen production. PT inhibits these fibrogenic pathways.
doi:10.1016/j.jss.2006.12.558
PMCID: PMC3571727  PMID: 17574589
biliary epithelium; cholangitis; fibrosis; gallstones
9.  The Epstein-Barr Virus Latent Membrane Protein 1 and Transforming Growth Factor–β1 Synergistically Induce Epithelial–Mesenchymal Transition in Lung Epithelial Cells 
The histopathology of idiopathic pulmonary fibrosis (IPF) includes the presence of myofibroblasts within so-called fibroblastic foci, and studies suggest that lung myofibroblasts may be derived from epithelial cells through epithelial–mesenchymal transition (EMT). Transforming growth factor (TGF)–β1 is expressed and/or activated in fibrogenesis, and induces EMT in lung epithelial cells in a dose-dependent manner. A higher occurrence of Epstein-Barr virus (EBV) has been reported in the lung tissue of patients with IPF. EBV expresses latent membrane protein (LMP) 1 during the latent phase of infection, and may play a role in the pathogenesis of pulmonary fibrosis inasmuch as LMP-1 may act as a constitutively active TNF-α receptor. Our data show a remarkable increase in mesenchymal cell markers, along with a concurrent reduction in the expression of epithelial cell markers in lung epithelial cells cotreated with LMP-1, and very low doses of TGF-β1. This effect was mirrored in lung epithelial cells infected with EBV expressing LMP1 and cotreated with TGF-β1. LMP1 pro-EMT signaling was identified, and occurs primarily through the nuclear factor–κB pathway and secondarily through the extracellular signal–regulated kinase (ERK) pathway. Activation of the ERK pathway was shown to be critical for aspects of TGF-β1–induced EMT. LMP1 accentuates the TGF-β1 activation of ERK. Together, these data demonstrate that the presence of EBV-LMP1 in lung epithelial cells synergizes with TGF-β1 to induce EMT. Our in vitro data may help to explain the observation that patients with IPF demonstrating positive staining for LMP1 in lung epithelial cells have a more rapid demise than patients in whom LMP1 is not detected.
doi:10.1165/rcmb.2009-0232OC
PMCID: PMC3135845  PMID: 20693406
latent membrane protein 1; transforming growth factor–β1; epithelial–mesenchymal transition; idiopathic pulmonary fibrosis; Epstein-Barr virus
10.  Secreted protein acidic and rich in cysteine (SPARC) is upregulated by transforming growth factor (TGF)-β and is required for TGF-β-induced hydrogen peroxide production in fibroblasts 
Background
Idiopathic pulmonary fibrosis (IPF) is a poorly understood progressive disease characterized by the recurrent damage of alveolar epithelial cells as well as inappropriate expansion and activation of fibroblasts resulting in pronounced extracellular matrix (ECM) deposition. Although recent studies have indicated the involvement of secreted protein acidic and rich in cysteine (SPARC), a matricellular protein regulating ECM deposition, in the pathogenesis of fibrosis, factors regulating SPARC expression or roles of SPARC in fibrosis have not been fully elucidated.
Results
Among the profibrotic factors examined in cultured fibroblasts, we showed that SPARC expression was upregulated mainly by transforming growth factor (TGF)-β. We also showed that expression of SPARC in the lung was upregulated in the murine bleomycin-induced pulmonary fibrosis model, which was inhibited by TGF-β receptor I inhibitor. Knockdown of SPARC in fibroblasts using siRNA or treatment with the antioxidant N-acetylcysteine attenuated epithelial cell injury induced by TGF-β-activated fibroblasts in a coculture system. We also demonstrated that SPARC was required for hydrogen peroxide (H2O2) production in fibroblasts treated with TGF-β. Furthermore, TGF-β activated integrin-linked kinase (ILK), which was inhibited by SPARC siRNA. Knockdown of ILK attenuated extracellular H2O2 generation in TGF-β-stimulated fibroblasts. Our results indicated that SPARC is upregulated by TGF-β and is required for TGF-β-induced H2O2 production via activation of ILK, and this H2O2 production from fibroblasts is capable of causing epithelial cell injury.
Conclusions
The results presented in this study suggest that SPARC plays a role in epithelial damage in the IPF lung via enhanced H2O2 production from fibroblasts activated by TGF-β. Therefore, SPARC inhibition may prevent epithelial injury in IPF lung and represent a potential therapeutic approach for IPF.
doi:10.1186/1755-1536-6-6
PMCID: PMC3610252  PMID: 23517551
SPARC; TGF-β; Hydrogen peroxide; Fibroblast; Pulmonary fibrosis
11.  Transforming Growth Factor-β Directly Induces p53-up-regulated Modulator of Apoptosis (PUMA) during the Rapid Induction of Apoptosis in Myc-driven B-cell Lymphomas* 
The Journal of Biological Chemistry  2012;288(7):5198-5209.
Background: TGF-β induces apoptosis in Burkitt's lymphoma cells.
Results: PUMA is a direct target gene of TGF-β signaling and is required for rapid apoptosis.
Conclusion: TGF-β-mediated direct induction of PUMA contributes to apoptosis in human and murine c-Myc-driven lymphomas.
Significance: These studies link TGF-β signaling and transcriptional activation of PUMA, two factors with critical roles in regulating B-cell survival.
c-Myc transformed human Burkitt's lymphoma (BL) cells are highly sensitive to TGF-β-induced apoptosis. Previously we demonstrated that TGF-β-mediated cell death in BL cells is regulated via the mitochondrial intrinsic apoptosis pathway, which is dependent on the activation of BAX and/or BAK. TGF-β directly induces transcription of the BH3-only protein BIK and represses expression of the pro-survival factor BCL-XL but has no effect on the direct BAX/BAK “activators” BIM or BID (tBID). Here we show that TGF-β induces the BH3-only activator PUMA to aid induction of the intrinsic cell death pathway. TGF-β also induced PUMA in normal germinal center CD77-positive centroblasts isolated from human tonsil tissue. PUMA was a direct TGF-β target gene in B-cells, and we identify a putative Smad-binding region within the human PUMA promoter that recruits Smad3 and Smad4 in cells in response to TGF-β signaling. Constitutive activity of the isolated Smad-binding region in luciferase reporter assays was dependent on Smad consensus sequences and was partially dependent on endogenous TGF-β signaling and Smad4. Knockdown of PUMA in BL cells using lentiviral shRNA resulted in slower kinetics of the TGF-β-mediated apoptotic response. Analysis of Eμ-Myc cell lines demonstrated that c-myc-driven murine lymphomas are also sensitive to TGF-β-mediated apoptosis. Moreover, Puma−/− Eμ-Myc lines demonstrated significantly delayed kinetics of the apoptotic response when compared with wild type lymphomas. TGF-β therefore induces a polygenic response in Myc-driven lymphomas involving transcription of PUMA, which is necessary for the rapid induction of cell death.
doi:10.1074/jbc.M112.410274
PMCID: PMC3576124  PMID: 23243310
Apoptosis; Bcl-2 Family Proteins; Lymphoma; Myc; Transforming Growth Factor Beta (TGFbeta); Burkitt's Lymphoma; PUMA
12.  Attenuation of CCl4-Induced Hepatic Fibrosis in Mice by Vaccinating against TGF-β1 
PLoS ONE  2013;8(12):e82190.
Transforming growth factor β1 (TGF-β1) is the pivotal pro-fibrogenic cytokine in hepatic fibrosis. Reducing the over-produced expression of TGF-β1 or blocking its signaling pathways is considered to be a promising therapeutic strategy for hepatic fibrosis. In this study, we evaluated the feasibility of attenuating hepatic fibrosis by vaccination against TGF-β1 with TGF-β1 kinoids. Two TGF-β1 kinoid vaccines were prepared by cross-linking TGF-β1-derived polypeptides (TGF-β125–[41-65] and TGF-β130–[83-112]) to keyhole limpet hemocyanin (KLH). Immunization with the two TGF-β1 kinoids efficiently elicited the production of high-levels of TGF-β1-specific antibodies against in BALB/c mice as tested by enzyme-linked immunosorbent assay (ELISA) and Western blotting. The antisera neutralized TGF-β1-induced growth-inhibition on mink lung epithelial cells (Mv1Lu) and attenuated TGF-β1-induced Smad2/3 phosphorylation, α-SMA, collagen type 1 alpha 2 (COL1A2), plasminogen activator inhibitor-1 (PAI-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) expression in the rat hepatic stellate cell (HSC) line, HSC-T6. Vaccination against TGF-β1 with the kinoids significantly suppressed CCl4-induced collagen deposition and the expression of α-SMA and desmin, attenuated hepatocyte apoptosis and accelerated hepatocyte proliferation in BALB/c mice. These results demonstrated that immunization with the TGF-β1 kinoids efficiently attenuated CCl4-induced hepatic fibrosis and liver injury. Our study suggests that vaccination against TGF-β1 might be developed into a feasible therapeutic approach for the treatment of chronic fibrotic liver diseases.
doi:10.1371/journal.pone.0082190
PMCID: PMC3859579  PMID: 24349218
13.  Quantitative analysis of transient and sustained transforming growth factor-β signaling dynamics 
Mathematical modeling and experimental analyses reveal that TGF-β ligand depletion has an important role in converting short-term graded signaling responses to long-term switch-like responses.
Cells respond in real time to the absolute number of TGF-β molecules in their environment.A single pulse of TGF-β stimulation results in transient SMAD activation whereas repeated short pulses of stimulation result in sustained SMAD activation.Ligand-induced short-term TGF-β/SMAD signaling activation is graded while long-term signaling response is switch-like or ultrasensitive.TGF-β ligand depletion is a major cause of conversion from graded short-term responses to ultrasensitive long-term responses.
The transforming growth factor-β (TGF-β) pathway is a prominent signaling pathway that regulates diverse aspects of cellular homeostasis, including proliferation, differentiation, migration, and death (Massague, 1998). Remarkably, the pleiotropic biological effects of TGF-β are mediated by a relatively simple signaling module (Clarke and Liu, 2008). An interesting question is how such an apparently straightforward and simple cascade can generate a wide array of biological responses depending on the cellular context.
Members of the TGF-β superfamily are frequently used as morphogens in early embryo development (Green, 2002). The best-studied examples include Dpp in Drosophila and Activin in Xenopus (Gurdon and Bourillot, 2001; Lander, 2007). In the developmental context, cells can respond to a graded ligand concentration and produce discrete biological responses (e.g., transcription of certain genes, proliferation, or differentiation; Green, 2002). To convert continuous morphogen stimulation into discrete responses, mechanisms must exist to provide a threshold for the cellular response. How variable TGF-β ligand doses quantitatively control intracellular signaling dynamics and how continuous ligand doses are translated into discontinuous cellular fate decisions remains poorly understood.
We have previously reported that ligand molecules per cell is the input variable to which the cells respond, and ligand number per cell is the best predictor of signaling responses (Zi and Klipp, 2007a; Clarke et al, 2009). Here, we developed an improved mathematical model to predict TGF-β signaling responses by calibrating the model with various experimental data sets from different TGF-β stimulations. Using a combined experimental and mathematical modeling approach, we showed that TGF-β pulse stimulation results in transient activation of the pathway while repeated short pulses at short time intervals lead to a sustained activation similar to persistent ligand exposure.
We next investigate the system response to variable doses of TGF-β in HaCaT cells. Our mathematical model predicts that the short-term Smad2 phosphorylation (after 45 min of TGF-β stimulation) is a graded response, while long-term Smad2 activation (after 24 h of TGF-β stimulation) is a switch-like response (Figure 5A and B). As shown in Figure 5A–D, both short- and long-term Smad2 phosphorylation can be saturated but doses of TGF-β that cause maximum response are quite different. Additionally, the shapes of response curves were different. The short-term Smad2 activation was a graded (Michaelis–Menten-like) response with a very low apparent Hill coefficient of about 0.8 (Figure 5A and C) while the long-term Smad2 activation (P-Smad2 at 24 h) yielded a switch-like response with an apparent Hill coefficient of about 4.5 (Figure 5B and D). Thus, the Smad2 response is initially graded and sharpens over time to become ultrasensitive. To address whether TGF-β-inducible gene expression responses are graded or switch-like in the short and long term, we measured mRNA levels of Smad7, an early responsive gene of TGF-β and protein levels of p21 and PAI-1 whose inductions are delayed and late, respectively. The experimental data show that Smad7 induction exhibits a graded response with corresponding Hill coefficients of about 1.3 (Figure 5E), which is consistent with the graded P-Smad2 response at 45 min (Figure 5A and C). PAI-1 induction in response to variable doses of TGF-β for 24 h is highly ultrasensitive with an apparent Hill coefficient of ∼5.3. Compared with Smad7 and PAI-1, p21 induction is only modest ultrasensitive (nHill≈2) (Figure 5G). These results suggest short-term gene induction by TGF-β appears to be graded while long-term targets are more switch-like. Finally, we measured the growth inhibitory response of HaCaT cells to variable doses of TGF-β. The level of BrdU incorporation is also ultrasensitive with an apparent Hill coefficient of about 4.3 (Figure 5H). Therefore, the long-term TGF-β growth inhibitory response also shows a switch-like behavior. Finally, we show that TGF-β depletion affects long-term Smad phosphorylation and switch-like response of TGF-β signaling system. These findings shed new light on how continuous ligand doses are translated into discontinuous cell fate decisions in biological systems.
In summary, we have shown that the dose and time course of TGF-β stimulation have profound effects on Smad signaling dynamics. The rate of ligand depletion controls the duration of Smad2 phosphorylation. Cells can respond to a short pulse of TGF-β stimulation, and periodic short ligand exposures are sufficient to generate long-term signaling responses. Short-term TGF-β stimulation causes only transient pathway activation and can be terminated by ligand depletion. TGF-β-induced Smad2 phosphorylation is graded in the short-term but ultrasensitive (switch-like) in the long-term (Figure 7). Additionally, cell growth arrest in response to TGF-β shows switch-like rather than graded behavior. Our modeling and experimental analyses suggest that ligand depletion is likely to be involved in sharpening a graded response into a switch-like response.
Mammalian cells can decode the concentration of extracellular transforming growth factor-β (TGF-β) and transduce this cue into appropriate cell fate decisions. How variable TGF-β ligand doses quantitatively control intracellular signaling dynamics and how continuous ligand doses are translated into discontinuous cellular fate decisions remain poorly understood. Using a combined experimental and mathematical modeling approach, we discovered that cells respond differently to continuous and pulsating TGF-β stimulation. The TGF-β pathway elicits a transient signaling response to a single pulse of TGF-β stimulation, whereas it is capable of integrating repeated pulses of ligand stimulation at short time interval, resulting in sustained phospho-Smad2 and transcriptional responses. Additionally, the TGF-β pathway displays different sensitivities to ligand doses at different time scales. While ligand-induced short-term Smad2 phosphorylation is graded, long-term Smad2 phosphorylation is switch-like to a small change in TGF-β levels. Correspondingly, the short-term Smad7 gene expression is graded, while long-term PAI-1 gene expression is switch-like, as is the long-term growth inhibitory response. Our results suggest that long-term switch-like signaling responses in the TGF-β pathway might be critical for cell fate determination.
doi:10.1038/msb.2011.22
PMCID: PMC3130555  PMID: 21613981
mathematical model; Smad; TGF-β; ultrasensitivity
14.  TGF-β autocrine pathway and MAPK signaling promote cell invasiveness and in vivo mammary adenocarcinoma tumor progression 
Oncology Reports  2012;28(2):567-575.
Breast cancer progression and metastasis have been linked to abnormal signaling by transforming growth factor-β (TGF-β) cytokines. In early-stage breast cancers, TGF-β exhibits tumor suppressor activity by repressing cell proliferation and inducing cell death, whereas in advanced-stage tumors, TGF-β promotes invasion and metastatic dissemination. The molecular mechanisms underlying pro-oncogenic activities of TGF-β are not fully understood. The present study validates the role of TGF-β signaling in cancer progression and explores mediators of pro-oncogenic TGF-β activities using the LM3 mammary adenocarcinoma cell line, derived from a spontaneous murine mammary adenocarcinoma. Expression of kinase-inactive TGF-β receptors decreased both basal and TGF-β-induced invasion. Analysis of signal transduction mediators showed that p38MAPK and MEK contribute to TGF-β stimulation of cell motility and invasion. TGF-β disrupted the epithelial actin structures supporting cell-cell adhesions, and increased linear actin filaments. Moreover, MEK and p38MAPK pathways showed opposite effects on actin remodeling in response to TGF-β. Blockade of Raf-MEK signaling enhanced TGF-β induction of actin stress-fibers whereas p38MAPK inhibitors blocked this effect. A novel observation was made that TGF-β rapidly activates the actin nucleation Arp2/3 complex. In addition, TGF-β stimulated matrix metalloproteinase MMP-9 secretion via a MAPK-independent pathway. Experiments using syngeneic mice showed that kinase-inactive TGF-β receptors inhibit the first stages of LM3 tumor growth in vivo. Our studies demonstrate that autocrine TGF-β signaling contributes to the invasive behavior of mammary carcinoma cells. Moreover, we show that both MAPK-dependent and -independent pathways are necessary for TGF-β-induced effects. Therefore, MEK-ERK and p38 MAPK pathways are potential venues for therapeutic intervention in pro-oncogenic TGF-β signaling.
doi:10.3892/or.2012.1813
PMCID: PMC3981025  PMID: 22614218
transforming growth factor-β; migration; invasion; matrix metalloproteinase-9; actin cytoskeleton; mammary adenocarcinoma
15.  TGF-β1 induces human alveolar epithelial to mesenchymal cell transition (EMT) 
Respiratory Research  2005;6(1):56.
Background
Fibroblastic foci are characteristic features in lung parenchyma of patients with idiopathic pulmonary fibrosis (IPF). They comprise aggregates of mesenchymal cells which underlie sites of unresolved epithelial injury and are associated with progression of fibrosis. However, the cellular origins of these mesenchymal phenotypes remain unclear. We examined whether the potent fibrogenic cytokine TGF-β1 could induce epithelial mesenchymal transition (EMT) in the human alveolar epithelial cell line, A549, and investigated the signaling pathway of TGF-β1-mediated EMT.
Methods
A549 cells were examined for evidence of EMT after treatment with TGF-β1. EMT was assessed by: morphology under phase-contrast microscopy; Western analysis of cell lysates for expression of mesenchymal phenotypic markers including fibronectin EDA (Fn-EDA), and expression of epithelial phenotypic markers including E-cadherin (E-cad). Markers of fibrogenesis, including collagens and connective tissue growth factor (CTGF) were also evaluated by measuring mRNA level using RT-PCR, and protein by immunofluorescence or Western blotting. Signaling pathways for EMT were characterized by Western analysis of cell lysates using monoclonal antibodies to detect phosphorylated Erk1/2 and Smad2 after TGF-β1 treatment in the presence or absence of MEK inhibitors. The role of Smad2 in TGF-β1-mediated EMT was investigated using siRNA.
Results
The data showed that TGF-β1, but not TNF-α or IL-1β, induced A549 cells with an alveolar epithelial type II cell phenotype to undergo EMT in a time-and concentration-dependent manner. The process of EMT was accompanied by morphological alteration and expression of the fibroblast phenotypic markers Fn-EDA and vimentin, concomitant with a downregulation of the epithelial phenotype marker E-cad. Furthermore, cells that had undergone EMT showed enhanced expression of markers of fibrogenesis including collagens type I and III and CTGF. MMP-2 expression was also evidenced. TGF-β1-induced EMT occurred through phosphorylation of Smad2 and was inhibited by Smad2 gene silencing; MEK inhibitors failed to attenuate either EMT-associated Smad2 phosphorylation or the observed phenotypic changes.
Conclusion
Our study shows that TGF-β1 induces A549 alveolar epithelial cells to undergo EMT via Smad2 activation. Our data support the concept of EMT in lung epithelial cells, and suggest the need for further studies to investigate the phenomenon.
doi:10.1186/1465-9921-6-56
PMCID: PMC1177991  PMID: 15946381
16.  CD4+CD25+FoxP3+ Regulatory Tregs inhibit fibrocyte recruitment and fibrosis via suppression of FGF-9 production in the TGF-β1 exposed murine lung 
Pulmonary fibrosis is a difficult to treat, often fatal disease whose pathogenesis involves dysregulated TGF-β1 signaling. CD4+CD25+FoxP3+ Regulatory T cells (“Tregs”) exert important effects on host tolerance and arise from naïve CD4+ lymphocytes in response to TGF-β1. However, the precise contribution of Tregs to experimentally induced murine lung fibrosis remains unclear. We sought to better understand the role of Tregs in this context. Using a model of fibrosis caused by lung specific, doxycycline inducible overexpression of the bioactive form of the human TGF-β1 gene we find that Tregs accumulate in the lung parenchyma within 5 days of transgene activation and that this enhancement persists to at least 14 days. Anti-CD25 Antibody mediated depletion of Tregs causes increased accumulation of soluble collagen and of intrapulmonary CD45+Col Iα1 fibrocytes. These effects are accompanied by enhanced local concentrations of the classical inflammatory mediators CD40L, TNF-α, and IL-1α, along with the neuroimmune molecule fibroblast growth factor 9 (FGF-9, also known as “glial activating factor”). FGF-9 expression localizes to parenchymal cells and alveolar macrophages in this model and antibody mediated neutralization of FGF-9 results in attenuated detection of intrapulmonary collagen and fibrocytes without affecting Treg quantities. These data indicate that CD4+CD25+FoxP3+ Tregs attenuate TGF-β1 induced lung fibrosis and fibrocyte accumulation in part via suppression of FGF-9.
doi:10.3389/fphar.2014.00080
PMCID: PMC4032896  PMID: 24904415
regulatory T cells; Fibrosis; TGF-β1; FGF-9; fibrocytes
17.  Role of Radiation-induced TGF-beta Signaling in Cancer Therapy 
TGF-β signaling regulates several different biological processes involving cell-growth, differentiation, apoptosis, motility, angiogenesis, epithelial mesenchymal transition and extracellular matrix production that affects embryonic development and pathogenesis of various diseases, including cancer, its effects depending on the cellular context and physiological environment. Growth suppression mediated by TGF-β signaling often associated with inhibition of c-myc, cdks and induction of p15, p27, Bax and p21. Despite its growth inhibitory effect, in certain conditions TGF-β may act as a promoter of cell proliferation and invasion. Loss of responsiveness to growth suppression by TGF-β due to mutation or loss of TGF-beta type II receptor (TβRII) and Smad4 in several different cancer cells are reported. In addition, TGF-β binding to its receptor activates many non-canonical signaling pathways. Radiation induced TGF-β is primarily involved in normal tissue injury and fibrosis. Seminal studies from our group have used radio-adjuvant therapies, involving classical components of the pathway such as TβRII and SMAD4 to overcome the growth promoting effects of TGF-β. The main impediment in the radiation-induced TGF-β signaling is the induction of SMAD7 that blocks TGF-β signaling in a negative feedback manner. It is well demonstrated from our studies that the use of neutralizing antibodies against TGF- β can render a robust radio-resistant effect. Thus, understanding the functional interactions of TGF-β signaling components of the pathway with other molecules may help tailor appropriate adjuvant radio-therapeutic strategies for treatment of solid tumors.
PMCID: PMC2844640  PMID: 20336170
TGF-β; Smads; Radiation; Cancer; Apoptosis
18.  TGF-β: Titan of Lung Fibrogenesis 
Current enzyme inhibition  2010;6(2):10.2174/10067.
Pulmonary fibrosis is characterized by epithelial cell injury, accumulation of myofibroblasts, and excessive deposition of collagen and other extracellular matrix elements, leading to loss of pulmonary function. Studies in both humans and animal models strongly suggest that TGF-β1 plays a pivotal role in the pathogenesis of pulmonary fibrosis. This review will first give an overview of TGF-β signaling and the effects of its inhibition on lung fibrogenesis. This overview includes information on TGF-β signal transduction pathways, the importance of TGF-β in the accumulation of myofibroblasts, the role of TGF-β in epithelial injury and apoptosis, the role of TGF-β in extracellular matrix remodeling, and the effects of inhibiting TGF-β signaling in animal models of lung fibrosis. Subsequently this review will highlight recent advances in two areas of particular interest to our research group: (1) TGF-β and proteoglycans; (2) TGF-β and histone deacetylases. Although our understanding of the role of TGF-β and its mechanisms of action in lung fibrogenesis has increased dramatically in recent years, there is still much to be learned about this important molecule, especially how TGF-β function is modulated in vivo, and its complex interactions with other factors expressed during lung injury and repair. Research in these areas will help identify novel therapeutic targets for the treatment of pulmonary fibrosis that will hopefully improve the prognosis of this devastating illness.
doi:10.2174/10067
PMCID: PMC3812949  PMID: 24187529
TGF-β; pulmonary fibrosis; myofibroblasts; epithelial-mesenchymal transition; apoptosis; integrin; reactive oxygen species; proteoglycan; sulf; histone deacetylase
19.  Angiotensin receptor blockade attenuates cigarette smoke–induced lung injury and rescues lung architecture in mice 
Chronic obstructive pulmonary disease (COPD) is a prevalent smoking-related disease for which no disease-altering therapies currently exist. As dysregulated TGF-β signaling associates with lung pathology in patients with COPD and in animal models of lung injury induced by chronic exposure to cigarette smoke (CS), we postulated that inhibiting TGF-β signaling would protect against CS-induced lung injury. We first confirmed that TGF-β signaling was induced in the lungs of mice chronically exposed to CS as well as in COPD patient samples. Importantly, key pathological features of smoking-associated lung disease in patients, e.g., alveolar injury with overt emphysema and airway epithelial hyperplasia with fibrosis, accompanied CS-induced alveolar cell apoptosis caused by enhanced TGF-β signaling in CS-exposed mice. Systemic administration of a TGF-β–specific neutralizing antibody normalized TGF-β signaling and alveolar cell death, conferring improved lung architecture and lung mechanics in CS-exposed mice. Use of losartan, an angiotensin receptor type 1 blocker used widely in the clinic and known to antagonize TGF-β signaling, also improved oxidative stress, inflammation, metalloprotease activation and elastin remodeling. These data support our hypothesis that inhibition of TGF-β signaling through angiotensin receptor blockade can attenuate CS-induced lung injury in an established murine model. More importantly, our findings provide a preclinical platform for the development of other TGF-β–targeted therapies for patients with COPD.
doi:10.1172/JCI46215
PMCID: PMC3248282  PMID: 22182843
20.  Inhibition of Integrin αvβ6, an Activator of Latent Transforming Growth Factor-β, Prevents Radiation-induced Lung Fibrosis 
Rationale: In experimental models, lung fibrosis is dependent on transforming growth factor (TGF)-β signaling. TGF-β is secreted in a latent complex with its propeptide, and TGF-β activators release TGF-β from this complex. Because the integrin αvβ6 is a major TGF-β activator in the lung, inhibition of αvβ6-mediated TGF-β activation is a logical strategy to treat lung fibrosis.
Objectives: To determine, by genetic and pharmacologic approaches, whether murine radiation-induced lung fibrosis is dependent on αvβ6.
Methods: Wild-type mice, αvβ6-deficient (Itgb6−/−) mice, and mice heterozygous for a Tgfb1 mutation that eliminates integrin-mediated activation (Tgfb1+/RGE) were exposed to 14 Gy thoracic radiation. Some mice were treated with an anti-αvβ6 monoclonal antibody or a soluble TGF-β receptor fusion protein. αvβ6 expression was determined by immunohistochemistry. Fibrosis, inflammation, and gene expression patterns were assessed 20–32 weeks postirradiation.
Measurements and Main Results: β6 Integrin expression increased within the alveolar epithelium 18 weeks postirradiation, just before onset of fibrosis. Itgb6−/− mice were completely protected from fibrosis, but not from late radiation-induced mortality. Anti-αvβ6 therapy (1–10 mg/kg/wk) prevented fibrosis, but only higher doses (6–10 mg/kg/wk) caused lung inflammation similar to that in Itgb6−/− mice. Tgfb1-haploinsufficient mice were also protected from fibrosis.
Conclusions: αvβ6-Mediated TGF-β activation is required for radiation-induced lung fibrosis. Together with previous data, our results demonstrate a robust requirement for αvβ6 in distinct fibrosis models. Inhibition of αvβ6-mediated TGF-β activation is a promising new approach for antifibrosis therapy.
doi:10.1164/rccm.200706-806OC
PMCID: PMC2176115  PMID: 17916808
inflammation; lymphocyte; monoclonal antibody
21.  Hypoxia-inducible Factor Prolyl-hydroxylase-2 Mediates Transforming Growth Factor Beta 1-induced Epithelial-mesenchymal Transition In Renal Tubular Cells 
Biochimica et biophysica acta  2013;1833(6):1454-1462.
Transforming growth factor beta 1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) in kidney epithelial cells plays a key role in renal tubulointerstitial fibrosis in chronic kidney diseases. As hypoxia-inducible factor (HIF)-1α is found to mediate TGF-β1-induced signaling pathway, we tested the hypothesis that HIF-1α and its upstream regulator prolyl hydroxylase domain-containing proteins (PHDs) are involved in TGF-β1-induced EMT using cultured renal tubular cells. Our results showed that TGF-β1 stimulated EMT in renal tubular cells as indicated by the significant decrease in epithelial marker P-cadherin, and the increase in mesenchymal markers α-smooth muscle actin (α-SMA) and fibroblast-specific protein 1 (FSP-1). Meanwhile, we found that TGF-β1 time-dependently increased HIF-1α and that HIF-1α siRNA significantly inhibited TGF-β1-induced EMT, suggesting that HIF-1α mediated TGF-β1 induced-EMT. Real-time PCR showed that PHD1 and PHD2, rather than PHD3, could be detected, with PHD2 as the predominant form of PHDs (PHD1:PHD2 = 0.21:1.0). Importantly, PHD2 mRNA and protein, but not PHD1, was decreased by TGF-β1. Furthermore, over-expression of PHD2 transgene almost fully prevented TGF-β1-induced HIF-1α accumulation and EMT marker changes, indicating that PHD2 is involved in TGF-β1-induced EMT. Finally, Smad2/3 inhibitor SB431542 prevented TGF-β1-induced PHD2 decrease, suggesting that Smad2/3 may mediate TGF-β1-induced EMT through PHD2/HIF-1α pathway. It is concluded that TGF-β1 decreased PHD2 expression via a Smad-dependent signaling pathway, thereby leading to HIF-1α accumulation and then EMT in renal tubular cells. The present study suggests that PHD2/HIF-1α is a novel signaling pathway mediating the fibrogenic effect of TGF-β1, and may be a new therapeutic target in chronic kidney diseases.
doi:10.1016/j.bbamcr.2013.02.029
PMCID: PMC3631109  PMID: 23466866
Prolyl hydroxylase domain-containing proteins; Smad signaling pathway; renal fibrosis
22.  The TGF-beta-Pseudoreceptor BAMBI is strongly expressed in COPD lungs and regulated by nontypeable Haemophilus influenzae 
Respiratory Research  2010;11(1):67.
Background
Nontypeable Haemophilus influenzae (NTHI) may play a role as an infectious trigger in the pathogenesis of chronic obstructive pulmonary disease (COPD). Few data are available regarding the influence of acute and persistent infection on tissue remodelling and repair factors such as transforming growth factor (TGF)-β.
Methods
NTHI infection in lung tissues obtained from COPD patients and controls was studied in vivo and using an in vitro model. Infection experiments were performed with two different clinical isolates. Detection of NTHI was done using in situ hybridization (ISH) in unstimulated and in in vitro infected lung tissue. For characterization of TGF-β signaling molecules a transcriptome array was performed. Expression of the TGF-pseudoreceptor BMP and Activin Membrane-bound Inhibitor (BAMBI) was analyzed using immunohistochemistry (IHC), ISH and PCR. CXC chemokine ligand (CXCL)-8, tumor necrosis factor (TNF)-α and TGF-β expression were evaluated in lung tissue and cell culture using ELISA.
Results
In 38% of COPD patients infection with NTHI was detected in vivo in contrast to 0% of controls (p < 0.05). Transcriptome arrays showed no significant changes of TGF-β receptors 1 and 2 and Smad-3 expression, whereas a strong expression of BAMBI with upregulation after in vitro infection of COPD lung tissue was demonstrated. BAMBI was expressed ubiquitously on alveolar macrophages (AM) and to a lesser degree on alveolar epithelial cells (AEC). Measurement of cytokine concentrations in lung tissue supernatants revealed a decreased expression of TGF-β (p < 0.05) in combination with a strong proinflammatory response (p < 0.01).
Conclusions
We show for the first time the expression of the TGF pseudoreceptor BAMBI in the human lung, which is upregulated in response to NTHI infection in COPD lung tissue in vivo and in vitro. The combination of NTHI-mediated induction of proinflammatory cytokines and inhibition of TGF-β expression may influence inflammation induced tissue remodeling.
doi:10.1186/1465-9921-11-67
PMCID: PMC2890648  PMID: 20513241
23.  TGF-β and NF-κB signal pathway cross-talk is mediated through TAK1 and SMAD7 in a subset of head and neck cancers 
Oncogene  2012;32(12):1549-1559.
TGF-β plays a dual role in epithelial malignancies, including head and neck squamous cell carcinoma (HNSCC). Attenuation of canonical TGF-β signaling enhances de novo tumor development, while TGF-β overexpression and signaling paradoxically promotes malignant progression. We recently observed that TGF-β-induced growth arrest response is attenuated, in association with aberrant activation of Nuclear Factor-κB (NF-κB), a transcription factor which promotes malignant progression in HNSCC. However, what role cross-talk between components of the TGF-β and NF-κB pathways plays in altered activation of these pathways has not been established. Here, we show TGF-β receptor II and TGF-β-activated kinase 1 (TAK1) are predominantly expressed in a subset of HNSCC tumors with nuclear activation of NF-κB family member RELA (p65). Further, TGF-β1 treatment induced sequential phosphorylation of TAK1, IKK, IκBα, and RELA in human HNSCC lines. TAK1 enhances TGF-β-induced NF-κB activation, as TAK1 siRNA knock-down decreased TGF-β1-induced phosphorylation of IKK, IκB, and RELA, degradation of IκBα, nuclear translocation, and DNA binding of RELA, and NF–κB-induced reporter and target gene transcription. Functionally, TAK1 siRNA inhibited cell proliferation, migration and invasion. Celastrol, a TAK1 inhibitor and anti-inflammatory used in traditional Chinese medicine, also decreased TGF-β1-induced phosphorylation of TAK1 and RELA, suppressed basal, TGF-β1- and TNFα-induced NF-κB reporter gene activity, and cell proliferation, while increasing sub-G0 DNA fragmentation and Annexin V markers of apoptosis. Furthermore, TGF-β and RELA activation promoted SMAD7 expression. In turn, SMAD7 preferentially suppressed TGF-β-induced SMAD and NF-κB reporters when compared with constitutive or TNF-α-induced NF-κB reporter gene activation. Thus, cross-talk by TGF-β via TAK1 and NF-κB promotes the malignant phenotype of HNSCC. Moreover, NF-κB may contribute to the downstream attenuation of canonical TGF-β signaling through increased SMAD7 expression. Celastrol highlights the therapeutic potential of agents targeting TAK1 as a key node in this pro-oncogenic TGF-β-NF-κB signal pathway.
doi:10.1038/onc.2012.171
PMCID: PMC3434281  PMID: 22641218
TAK1; SMAD7; TGF-β; NF-κB; celastrol; head and neck cancer
24.  Epithelium-specific deletion of TGF-β receptor type II protects mice from bleomycin-induced pulmonary fibrosis 
Idiopathic pulmonary fibrosis (IPF) is a chronic fibroproliferative pulmonary disorder for which there are currently no treatments. Although the etiology of IPF is unknown, dysregulated TGF-β signaling has been implicated in its pathogenesis. Recent studies also suggest a central role for abnormal epithelial repair. In this study, we sought to elucidate the function of epithelial TGF-β signaling via TGF-β receptor II (TβRII) and its contribution to fibrosis by generating mice in which TβRII was specifically inactivated in mouse lung epithelium. These mice, which are referred to herein as TβRIINkx2.1-cre mice, were used to determine the impact of TβRII inactivation on (a) embryonic lung morphogenesis in vivo; and (b) the epithelial cell response to TGF-β signaling in vitro and in a bleomycin-induced, TGF-β–mediated mouse model of pulmonary fibrosis. Although postnatally viable with no discernible abnormalities in lung morphogenesis and epithelial cell differentiation, TβRIINkx2.1-cre mice developed emphysema, suggesting a requirement for epithelial TβRII in alveolar homeostasis. Absence of TβRII increased phosphorylation of Smad2 and decreased, but did not entirely block, phosphorylation of Smad3 in response to endogenous/physiologic TGF-β. However, TβRIINkx2.1-cre mice exhibited increased survival and resistance to bleomycin-induced pulmonary fibrosis. To our knowledge, these findings are the first to demonstrate a specific role for TGF-β signaling in the lung epithelium in the pathogenesis of pulmonary fibrosis.
doi:10.1172/JCI42090
PMCID: PMC3007138  PMID: 21135509
25.  Transforming Growth Factor-β1 Downregulates Vascular Endothelial Growth Factor-D Expression in Human Lung Fibroblasts via the Jun NH2-Terminal Kinase Signaling Pathway 
Molecular Medicine  2014;20(1):120-134.
Vascular endothelial growth factor (VEGF)-D, a member of the VEGF family, induces both angiogenesis and lymphangiogenesis by activating VEGF receptor-2 (VEGFR-2) and VEGFR-3 on the surface of endothelial cells. Transforming growth factor (TGF)-β1 has been shown to stimulate VEGF-A expression in human lung fibroblast via the Smad3 signaling pathway and to induce VEGF-C in human proximal tubular epithelial cells. However, the effects of TGF-β1 on VEGF-D regulation are unknown. To investigate the regulation of VEGF-D, human lung fibroblasts were studied under pro-fibrotic conditions in vitro and in idiopathic pulmonary fibrosis (IPF) lung tissue. We demonstrate that TGF-β1 downregulates VEGF-D expression in a dose- and time-dependent manner in human lung fibroblasts. This TGF-β1 effect can be abolished by inhibitors of TGF-β type I receptor kinase and Jun NH2-terminal kinase (JNK), but not by Smad3 knockdown. In addition, VEGF-D knockdown in human lung fibroblasts induces G1/S transition and promotes cell proliferation. Importantly, VEGF-D protein expression is decreased in lung homogenates from IPF patients compared with control lung. In IPF lung sections, fibroblastic foci show very weak VEGF-D immunoreactivity, whereas VEGF-D is abundantly expressed within alveolar interstitial cells in control lung. Taken together, our data identify a novel mechanism for downstream signal transduction induced by TGF-β1 in lung fibroblasts, through which they may mediate tissue remodeling in IPF.
doi:10.2119/molmed.2013.00123
PMCID: PMC3960396  PMID: 24515257

Results 1-25 (1117631)