Balancing the activation state of the endothelium via two distinct TGF-beta type I receptors

The generation of mice lacking specific components of the transforming growth factor-beta (TGF-beta) signal tranduction pathway shows that TGF-beta is a key player in the development and physiology of the cardiovascular system. Both pro- and anti-angiogenic properties have been ascribed to TGF-beta, for which the molecular mechanisms are unclear. Here we report that TGF-beta can activate two distinct type I receptor/Smad signalling pathways with opposite effects. TGF-beta induces phosphorylation of Smad1/5 and Smad2 in endothelial cells and these effects can be blocked upon selective inhibition of ALK1 or ALK5 expression, respectively. Whereas the TGF-beta/ALK5 pathway leads to inhibition of cell migration and proliferation, the TGF-beta/ALK1 pathway induces endothelial cell migration and proliferation. We identified genes that are induced specifically by TGF-beta-mediated ALK1 or ALK5 activation. Id1 was found to mediate the TGF-beta/ALK1-induced (and Smad-dependent) migration, while induction of plasminogen activator inhibitor-1 by activated ALK5 may contribute to the TGF-beta-induced maturation of blood vessels. Our results suggest that TGF-beta regulates the activation state of the endothelium via a fine balance between ALK5 and ALK1 signalling.     

Negative regulation of inducible nitric-oxide synthase expression mediated through transforming growth factor-beta-dependent modulation of transcription factor TCF11

Inducible nitric-oxide synthase (iNOS) plays a central role in the regulation of vascular function and response to injury. A central mediator controlling iNOS expression is transforming growth factor-beta (TGF-beta), which represses its expression through a mechanism that is poorly understood. We have identified a binding site in the iNOS promoter that interacts with the nuclear heterodimer TCF11/MafG using chromatin immunoprecipitation and mutation analyses. We demonstrate that binding at this site acts to repress the induction of iNOS gene expression by cytokines. We show that this repressor is induced by TGF-beta1 and by Smad6-short, which enhances TGF-beta signaling. In contrast, the up-regulation of TCF11/MafG binding could be suppressed by overexpression of the TGF-beta inhibitor Smad7, and a small interfering RNA to TCF11 blocked the suppression of iNOS by TGF-beta. The binding of TCF11/MafG to the iNOS promoter could be enhanced by phorbol 12-myristate 13-acetate and suppressed by the protein kinase C inhibitor staurosporine. Moreover, the induction of TCF11/MafG binding by TGF-beta and Smad6-short could be blocked by staurosporine, and the effect of TGF-beta was blocked by the selective protein kinase C inhibitor calphostin C. Consistent with the in vitro data, we found suppression of TCF11 coincident with iNOS up-regulation in a rat model of endotoxemia, and we observed a highly significant negative correlation between TCF11 and nitric oxide production. Furthermore, treatment with activated protein C, a serine protease effective in septic shock, blocked the down-regulation of TCF11 and suppressed endotoxin-induced iNOS. Overall, our results demonstrate a novel mechanism by which iNOS expression is regulated in the context of inflammatory activation.     

Transcriptional induction of Smurf2 ubiquitin ligase by TGF-beta

Smad ubiquitination regulatory factor 2 (Smurf2), a ubiquitin ligase for Smads, plays critical roles in the regulation of transforming growth factor-beta (TGF-beta)-Smad signaling via ubiquitin-dependent degradation of Smad2 and Smad7. We found that TGF-beta stimulates Smurf2 expression. TGF-beta activated the Smurf2 promoter in a TGF-beta responsive cell lines, whereas IL-1alpha, PDGF and epidermal growth factor did not. TGF-beta-mediated Smurf2 promoter activation was inhibited by Smad7 or an activin receptor-like kinase 5 inhibitor but not by dominant negative Smad or disruption of Smad-binding elements in the promoter. Moreover, inhibition of the phosphatidil inositol 3 kinase (PI3K)/Akt pathway suppressed TGF-beta-mediated Smurf2 induction. These results suggest that TGF-beta stimulates Smurf2 expression by Smad-independent pathway such as PI3K/Akt pathway via TGF-beta receptor.     

Platelet factor 4 inhibits thrombomodulin-dependent activation of thrombin-activatable fibrinolysis inhibitor (TAFI) by thrombin

Thrombomodulin (TM) is a cofactor for thrombin-mediated activation of protein C and thrombin-activatable fibrinolysis inhibitor (TAFI) and thereby helps coordinate coagulation, anticoagulation, fibrinolysis, and inflammation. Platelet factor 4 (PF4), a platelet α-granule protein and a soluble cofactor for TM-dependent protein C activation, stimulates protein C activation in vitro and in vivo. In contrast to stimulation of protein C activation, PF4 is shown here to inhibit activation of TAFI by thrombin-TM. Consequences of inhibition of TAFI activation by PF4 included loss of TM-dependent prolongation of clot lysis times in hemophilia A plasma and loss of TM-stimulated conversion of bradykinin (BK) to des-Arg(9)-BK by TAFIa in normal plasma. Thus, PF4 modulates the substrate specificity of the thrombin-TM complex by selectively enhancing protein C activation while inhibiting TAFI activation, thereby preventing the generation of the antifibrinolytic and anti-inflammatory activities of TAFIa. To block the inhibitory effects of PF4 on TAFI activation, heparin derivatives were tested for their ability to retain high affinity binding to PF4 despite having greatly diminished anticoagulant activity. N-acetylated heparin (NAc-Hep) lacked detectable anticoagulant activity in activated partial thromboplastin time clotting assays but retained high affinity binding to PF4 and effectively reversed PF4 binding to immobilized TM. NAc-Hep permitted BK conversion to des-Arg(9)-BK by TAFIa in the presence of PF4. In a clot lysis assay on TM-expressing cells using hemophilia A plasma, NAc-Hep prevented PF4-mediated inhibition of TAFI activation and the antifibrinolytic functions of TAFIa. Accordingly, NAc-Hep or similar heparin derivatives might provide therapeutic benefits by diminishing bleeding complications in hemophilia A via restoration of TAFIa-mediated protection of clots against premature lysis.     

Transforming growth factor-beta1 inhibition of vascular smooth muscle cell activation is mediated via Smad3

Activation of vascular smooth muscle cells (VSMCs) by proinflammatory cytokines is a key feature of atherosclerotic lesion formation. Transforming growth factor (TGF)-beta1 is a pleiotropic growth factor that can modulate the inflammatory response in diverse cell types including VSMCs. However, the mechanisms by which TGF-beta1 is able to mediate these effects remains incompletely understood. We demonstrate here that the ability of TGF-beta1 to inhibit markers of VSMC activation, inducible nitric-oxide synthase (iNOS) and interleukin (IL)-6, is mediated through its downstream effector Smad3. In reporter gene transfection studies, we found that among a panel of Smads, Smad3 could inhibit iNOS induction in an analogous manner as exogenous TGF-beta1. Adenoviral overexpression of Smad3 potently repressed inducible expression of endogenous iNOS and IL-6. Conversely, TGF-beta1 inhibition of cytokine-mediated induction of iNOS and IL-6 expression was completely blocked in Smad3-deficient VSMCs. Previous studies demonstrate that CCAAT/enhancer-binding protein (C/EBP) and NF-kappaB sites are critical for cytokine induction of both the iNOS and IL-6 promoters. We demonstrate that the inhibitory effect of Smad3 occurs via a novel antagonistic effect of Smad3 on C/EBP DNA-protein binding and activity. Smad3 mediates this effect in part by inhibiting C/EBP-beta and C/EBP-delta through distinct mechanisms. Furthermore, we find that Smad3 prevents the cooperative induction of the iNOS promoter by C/EBP and NF-kappaB. These data demonstrate that Smad3 plays an essential role in mediating TGF-beta1 anti-inflammatory response in VSMCs.     

Smad7 and Smad6 differentially modulate transforming growth factor beta -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.     

Disruption of transforming growth factor-beta signaling by curcumin induces gene expression of peroxisome proliferator-activated receptor-gamma in rat hepatic stellate cells

Activation of hepatic stellate cells (HSC), the major effectors of hepatic fibrogenesis, is coupled with sequential alterations in gene expression, including an increase in receptors for transforming growth factor-beta (TGF-beta) and a dramatic reduction in the peroxisome proliferator-activated receptor-gamma (PPAR-gamma). The relationship between them remains obscure. We previously demonstrated that curcumin induced gene expression of PPAR-gamma in activated HSC, leading to reducing cell proliferation, inducing apoptosis and suppressing expression of extracellular matrix genes. The underlying molecular mechanisms are largely unknown. We recently observed that stimulation of PPAR-gamma activation suppressed gene expression of TGF-beta receptors in activated HSC, leading to the interruption of TGF-beta signaling. This observation supported our assumption of an antagonistic relationship between PPAR-gamma activation and TGF-beta signaling in HSC. In this study, we further hypothesize that TGF-beta signaling might negatively regulate gene expression of PPAR-gamma in activated HSC. The present report demonstrates that exogenous TGF-beta1 inhibits gene expression of PPAR-gamma in activated HSC, which is eliminated by the pretreatment with curcumin likely by interrupting TGF-beta signaling. Transfection assays further indicate that blocking TGF-beta signaling by dominant negative type II TGF-beta receptor increases the promoter activity of PPAR-gamma gene. Promoter deletion assays, site-directed mutageneses, and gel shift assays localize two Smad binding elements (SBEs) in the PPAR-gamma gene promoter, acting as curcumin response elements and negatively regulating the promoter activity in passaged HSC. The Smad3/4 protein complex specifically binds to the SBEs. Overexpression of Smad4 dose dependently eliminates the inhibitory effects of curcumin on the PPAR-gamma gene promoter and TGF-beta signaling. Taken together, these results demonstrate that the interruption of TGF-beta signaling by curcumin induces gene expression of PPAR-gamma in activated HSC in vitro. Our studies provide novel insights into the molecular mechanisms of curcumin in the induction of PPAR-gamma gene expression and in the inhibition of HSC activation.     

Ectopic expression of Smad7 inhibits transforming growth factor-beta responses in vascular smooth muscle cells.

Vascular injury stimulates the cytokine-growth factor network in the vascular wall, including transforming growth factor-beta (TGF-beta). Reportedly, the intracellular signaling of TGF-beta is mediated by Smad proteins. We tested the effects of the ectopic expression of inhibitory Smads in cultured rat smooth muscle cells (SMC) to identify the role of TGF-beta/Smad signaling on the phenotypic modulation of SMC. The cells exposed to human recombinant TGF-beta1 (10 ng/ml) were stimulated Smad2 phosphorylation. Infection with the replication-deficient adenovirus vector expressing Smad7, but not bacterial beta-galactosidase or Smad6, was found to inhibit TGF-beta-induced Smad2 phosphorylation in a dose-dependent manner. TGF-beta suppressed the serum-induced proliferation of SMC from 36.3% to 51.0% (p<0.01), as measured by hand-counting, and this inhibition was attenuated by the ectopic expression of Smad7 (from 30.7% to 74.8% of the reduction of TGF-beta-response, p<0.05), but not Smad6. A BrdU incorporation assay also showed that TGF-beta-mediated growth inhibition was attenuated by exogenous Smad7 and that this inhibition can be reversed by an additional expression of exogenous Smad2. TGF-beta increased the expression of alpha-smooth muscle actin and myosin heavy chain by 1.3-fold and 1.6-fold in comparison to the control, respectively, and these increases were attenuated by exogenous Smad7, but not Smad6. Our data indicate that Smads mediate TGF-beta responses on SMC phenotypes. Smad7, but not Smad6, may specifically act as an inhibitor of TGF-beta responses.     

Retracted Article : Gonadotropin releasing hormone analogue (GnRHa) alters the expression and activation of Smad in human endometrial epithelial and stromal cells

Gonadotropin releasing hormone analogues (GnRHa) are often used to regress endometriosis implants and prevent premature luteinizing hormone surges in women undergoing controlled ovarian stimulation. In addition to GnRH central action, the expression of GnRH and receptors in the endometrium implies an autocrine/paracrine role for GnRH and an additional site of action for GnRHa. To further examine the direct action of GnRH (Leuprolide acetate) in the endometrium, we determined the effect of GnRH on endometrial stromal (ESC) and endometrial surface epithelial (HES) cells expression and activation of Smads (Smad3, -4 and -7), intracellular signals activated by transforming growth factor beta (TGF-beta), a key cytokine expressed in the endometrium. The results show that GnRH (0.1 microM) increased the expression of inhibitory Smad7 mRNA in HES with a limited effect on ESC, while moderately increasing the common Smad4 and Smad7 protein levels in these cells (P < 0.05). GnRH in a dose- (0.01 to 10 microM) and time- (5 to 30 min) dependent manner decreased the rate of Smad3 activation (phospho-Smad3, pSmad3), and altered Smad3 cellular distribution in both cell types. Pretreatment with Antide (GnRH antagonist) resulted in further suppression of Smad3 induced by GnRH, with Antide inhibition of pSmad3 in ESC. Furthermore, co-treatment of the cells with GnRH + TGF-beta, or pretreatment with TGF-beta type II receptor antisense to block TGF-beta autocrine/paracrine action, in part inhibited TGF-beta activated Smad3. In conclusion, the results indicate that GnRH acts directly on the endometrial cells altering the expression and activation of Smads, a mechanism that could lead to interruption of TGF-beta receptor signaling mediated through this pathway in the endometrium.