Internalization-dependent and -independent requirements for transforming growth factor beta receptor signaling via the Smad pathway

Members of the transforming growth factor β (TGF-β) family of proteins signal through cell surface transmembrane serine/threonine protein kinases known as type I and type II receptors. The TGF-β signal is extended through phosphorylation of receptor-associated Smad proteins by the type I receptor. Although numerous investigations have established the sequence of events in TGF-β receptor (TGF-βR) activation, none have examined the role of the endocytic pathway in initiation and/or maintenance of the signaling response. In this study we investigated whether TGF-βR internalization modulates type I receptor activation, the formation of a functional receptor/Smad/SARA complex, Smad2/3 phosphorylation or nuclear translocation, and TGF-β-dependent reporter gene activity. Our data provide evidence that, whereas type I receptor phosphorylation and association of SARA and Smad2 with the TGF-βR complex take place independently of clathrin lattice formation, Smad2 or Smad3 activation and downstream signaling only occur after endocytic vesicle formation. Thus, TGF-βR endocytosis is not simply a way to dampen the signaling response but instead is required to propagate signaling via the Smad pathway.     

Modulation of transforming growth factor beta signalling pathway genes by transforming growth factor beta in human osteoarthritic chondrocytes: involvement of Sp1 in both early and late response cells to transforming growth factor beta

Introduction  

Transforming growth factor beta (TGFβ) plays a central role in morphogenesis, growth, and cell differentiation. This cytokine is particularly important in cartilage where it regulates cell proliferation and extracellular matrix synthesis. While the action of TGFβ on chondrocyte metabolism has been extensively catalogued, the modulation of specific genes that function as mediators of TGFβ signalling is poorly defined. In the current study, elements of the Smad component of the TGFβ intracellular signalling system and TGFβ receptors were characterised in human chondrocytes upon TGFβ1 treatment.     

The Smad pathway in transforming growth factor-β signaling

The transforming growth factor b (TGF-b) superfamily comprises a great number of structurally related polypeptide growth factors, such as TGF-bs, activins, inhibins, bone morphogenic proteins (BMPs), growth differentiation factors (GDFs), M黮lerian inhibitory substance, and glial cell-derived neurotrophic factor (GDNF), etc[1]. The TGF-b superfamily members are multifunctional agonists involved in a broad spectrum of biological processes such as cell proliferation and differentiation, e…     

Cell-type-specific activation of PAK2 by transforming growth factor beta independent of Smad2 and Smad3

Transforming growth factor beta (TGF-beta) causes growth arrest in epithelial cells and proliferation and morphological transformation in fibroblasts. Despite the ability of TGF-beta to induce various cellular phenotypes, few discernible differences in TGF-beta signaling between cell types have been reported, with the only well-characterized pathway (the Smad cascade) seemingly under identical control. We determined that TGF-beta receptor signaling activates the STE20 homolog PAK2 in mammalian cells. PAK2 activation occurs in fibroblast but not epithelial cell cultures and is independent of Smad2 and/or Smad3. Furthermore, we show that TGF-beta-stimulated PAK2 activity is regulated by Rac1 and Cdc42 and dominant negative PAK2 or morpholino antisense oligonucleotides to PAK2 prevent the morphological alteration observed following TGF-beta addition. Thus, PAK2 represents a novel Smad-independent pathway that differentiates TGF-beta signaling in fibroblast (growth-stimulated) and epithelial cell (growth-inhibited) cultures.     

Requirement of Ras/MAPK pathway activation by transforming growth factor beta for transforming growth factor beta 1 production in a Smad-dependent pathway

Our previous results have shown that transforming growth factor beta (TGFbeta) rapidly activates Ras, as well as both ERKs and SAPKs. In order to address the biological significance of the activation of these pathways by TGFbeta, here we examined the role of the Ras/MAPK pathways and the Smads in TGFbeta(3) induction of TGFbeta(1) expression in untransformed lung and intestinal epithelial cells. Expression of either a dominant-negative mutant of Ras (RasN17) or a dominant-negative mutant of MKK4 (DN MKK4), or addition of the MEK1 inhibitor PD98059, inhibited the ability of TGFbeta(3) to induce AP-1 complex formation at the TGFbeta(1) promoter, and the subsequent induction of TGFbeta(1) mRNA. The primary components present in this TGFbeta(3)-inducible AP-1 complex at the TGFbeta(1) promoter were JunD and Fra-2, although c-Jun and FosB were also involved. Furthermore, deletion of the AP-1 site in the TGFbeta(1) promoter or addition of PD98059 inhibited the ability of TGFbeta(3) to stimulate TGFbeta(1) promoter activity. Collectively, our data demonstrate that TGFbeta(3) induction of TGFbeta(1) is mediated through a signaling cascade consisting of Ras, the MAPKKs MKK4 and MEK1, the MAPKs SAPKs and ERKs, and the specific AP-1 proteins Fra-2 and JunD. Although Smad3 and Smad4 were not detectable in TGFbeta(3)-inducible AP-1 complexes at the TGFbeta(1) promoter, stable expression of dominant-negative Smad3 could significantly inhibit the ability of TGFbeta(3) to stimulate TGFbeta(1) promoter activity. Transient expression of dominant-negative Smad4 also inhibited the ability of TGFbeta(3) to transactivate the TGFbeta(1) promoter. Thus, although the Ras/MAPK pathways are essential for TGFbeta(3) induction of TGFbeta(1), Smads may only contribute to this biological response in an indirect manner.     

Expression of connective tissue growth factor, a biomarker in senescence of human diploid fibroblasts, is up-regulated by a transforming growth factor-beta-mediated signaling pathway

Molecular changes associated with cellular senescence in human diploid fibroblasts (HDF), IMR-90, were analyzed by two-dimensional differential proteome analysis. A high percentage of replicative senescent cells were positive for senescence-associated beta-galactosidase activity, and displayed elevated levels of p21 and p53 proteins. Comparison of early population doubling level (PDL) versus replicative senescent cells among the 1000 spots resolved on gels revealed that the signal intensities of six spots were increased fivefold, whereas those of four spots were decreased. Proteome analysis data demonstrated that connective tissue growth factor (CTGF) is an age-associated protein. Up-regulation of CTGF expression in senescent cells was further confirmed by Western blotting and RT-PCR. We postulate that CTGF expression is controlled, in part, by transforming growth factor-beta (TGF-beta), in view of the high levels of TGF-beta isoforms as well as type I and II receptors detected only in late PDL of HDF cells. To verify this hypothesis, we stimulated early PDL cells with TGF-beta1 as well as stress inducing agents such as hydrogen peroxide. As expected, CTGF expression and Smad protein phosphorylation were dramatically increased up to observed levels in normal replicative senescent cells. In vivo experiments disclosed that CTGF, pSmad, and p53 were constitutively expressed at basal levels in up to 18-month-old rat liver, and expression was significantly up-regulated in 24-month-old rat tissue. However, expression patterns were not altered at all periods examined in livers of caloric-restricted rats. In view of both in vitro and in vivo data, we propose that the TGF-beta/Smad pathway functions in the induction of CTGF, a novel biomarker protein of cellular senescence in human fibroblasts.     

Divergence of epidermal growth factor - transforming growth factor beta signaling in embryonic orofacial tissue

The epidermal growth factor (EGF) and transforming growth factor beta (TGFbeta) families of signaling molecules play a major role in growth and development of embryos. Abrogation of either signaling pathway results in defects in embryogenesis, including cleft palate. In the developing palate, both EGF and TGFbeta regulate cellular proliferation, extracellular matrix synthesis, and cellular differentiation but often in an opposing manner. Evidence from various adult cell types suggests the existence of cross talk between the EGF and TGFbeta signaling pathways, although it is unclear whether such cross talk exists in murine embryonic maxillary mesenchymal cells, from which the developing palate is derived. In this study, embryonic maxillary mesenchymal cells in culture were treated with EGF and TGFbeta, either singly or in combination, and the cells were subsequently examined for signaling interactions between these two pathways. Immunoblot analyses of nuclear extracts of embryonic maxillary mesenchymal cells revealed that TGFbeta-induced nuclear translocation of Smad 2 and Smad 3 proteins was not affected by EGF. Conversely, immunoblot analyses of whole-cell extracts of these cells indicated that EGF-induced phosphorylation of extracellular signal-regulated kinase proteins, ERK1 and ERK2, was not affected by TGFbeta. Expression of a transfected luciferase reporter gene driven by a promoter with Smad binding elements was induced by TGFbeta in these cells but was not affected by EGF. Last, TGFbeta was found to induce expression of the endogenous gelatinase B gene in embryonic maxillary mesenchymal cells; however, this effect was independent of any interaction of EGF. Collectively, data from this study suggest that the EGF and TGFbeta signal transduction pathways do not converge in murine embryonic maxillary mesenchymal cells.     

Plasma transforming growth factor beta1, metalloproteinase-1 and tissue inhibitor of metalloproteinases-1 in acute viral hepatitis type B

AIM: Antiproliferative, pro-apoptotic and immunosuppressive activity effects suggest crucial role of transforming growth factor (TGF)-beta1, metalloproteinase (MMP)-1 and its tissue inhibitor (TIMP)-1 in the pathogenesis of acute liver injury that in some patients precede development of chronic liver diseases and fibrogenesis. The aim of this study was to evaluate effect of acute HBV infection on plasma TGF-beta1, MMP-1 and TIMP-1 levels. METHODS: TGF-beta1, MMP-1 and TIMP-1 plasma concentrations were measured with an enzyme immunoassay in 39 patients with acute viral hepatitis type B. Baseline measurement was performed within the first week of jaundice and then weekly up to the fourth week of the disease. Results were compared to baseline and normal values and to liver function tests. RESULTS: Plasma concentrations of TGF-beta1, TIMP-1 and MMP-1 were significantly elevated in the first week of acute viral B hepatitis in comparison to normal. Analysis of individual values demonstrated significant positive correlation between plasma concentrations of TGF-beta1 and TIMP-1. There was no correlation between MMP-1 and TGF-beta1 or TIMP-1. Significant correlation was demonstrated between both TGF-beta1 and ALT or AST as well as between TIMP-1 and ALT, AST or bilirubin. Elevated baseline levels of both TGF-beta1 and TIMP-1 decreased gradually in consecutive weeks of the disease. TGF-beta1 but not TIMP-1 plasma concentrations were significantly lower in 3rd and 4th week than baseline values. MMP-1 concentration remained on baseline level in the 2nd week of the disease. However in the 3rd week its values increased suddenly but the significant difference in comparison to baseline was observed only in 4th week. CONCLUSIONS: These results indicate important role of TGF-beta1, TIMP-1 and MMP-1 in acute viral hepatitis, that seems to be connected first of all with hepatocytes damage. Their role in extracellular matrix metabolism during acute liver injury needs further evaluation.     

MicroRNA-155 is regulated by the transforming growth factor beta/Smad pathway and contributes to epithelial cell plasticity by targeting RhoA

Transforming growth factor β (TGF-β) signaling facilitates metastasis in advanced malignancy. While a number of protein-encoding genes are known to be involved in this process, information on the role of microRNAs (miRNAs) in TGF-β-induced cell migration and invasion is still limited. By hybridizing a 515-miRNA oligonucleotide-based microarray library, a total of 28 miRNAs were found to be significantly deregulated in TGF-β-treated normal murine mammary gland (NMuMG) epithelial cells but not Smad4 knockdown NMuMG cells. Among upregulated miRNAs, miR-155 was the most significantly elevated miRNA. TGF-β induces miR-155 expression and promoter activity through Smad4. The knockdown of miR-155 suppressed TGF-β-induced epithelial-mesenchymal transition (EMT) and tight junction dissolution, as well as cell migration and invasion. Further, the ectopic expression of miR-155 reduced RhoA protein and disrupted tight junction formation. Reintroducing RhoA cDNA without the 3′ untranslated region largely reversed the phenotype induced by miR-155 and TGF-β. In addition, elevated levels of miR-155 were frequently detected in invasive breast cancer tissues. These data suggest that miR-155 may play an important role in TGF-β-induced EMT and cell migration and invasion by targeting RhoA and indicate that it is a potential therapeutic target for breast cancer intervention.     

Smad7 antagonizes transforming growth factor beta signaling in the nucleus by interfering with functional Smad-DNA complex formation

Smad7 plays an essential role in the negative-feedback regulation of transforming growth factor beta (TGF-beta) signaling by inhibiting TGF-beta signaling at the receptor level. It can interfere with binding to type I receptors and thus activation of receptor-regulated Smads or recruit the E3 ubiquitin ligase Smurf to receptors and thus target them for degradation. Here, we report that Smad7 is predominantly localized in the nucleus of Hep3B cells. The targeted expression of Smad7 in the nucleus conferred superior inhibitory activity on TGF-beta signaling, as determined by reporter assay in mammalian cells and by its effect on zebrafish embryogenesis. Furthermore, Smad7 repressed Smad3/4-, Smad2/4-, and Smad1/4-enhanced reporter gene expression, indicating that Smad7 can function independently of type I receptors. An oligonucleotide precipitation assay revealed that Smad7 can specifically bind to the Smad-responsive element via its MH2 domain, and DNA-binding activity was further confirmed in vivo with the promoter of PAI-1, a TGF-beta target gene, by chromatin immunoprecipitation. Finally, we provide evidence that Smad7 disrupts the formation of the TGF-beta-induced functional Smad-DNA complex. Our findings suggest that Smad7 inhibits TGF-beta signaling in the nucleus by a novel mechanism.