Negative Regulator Of TGF-beta Signaling Research Articles

Convergence of Clinical and Cellular Phenotypes Among Patients with STAT3 and ERBB2IP Mutations

A number of congenital syndromes with connective tissue abnormalities are also associated with allergic disease, some of which are caused by altered TGF-beta signaling. Patients with loss of STAT3 function share multiple clinical features with these syndromes. We therefore sought to identify common cellular changes among patients with heterozygous loss-of-function ERBB2IP mutations, a negative regulator of TGF-beta signaling, and patients with dominant negative STAT3 mutations.

97 : Silencing MicroRNA-21 ameliorates Th17 mediated autoimmune inflammation

IL-17 producing Th17 cells are the key participants in various autoimmune diseases including multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Although differentiation of Th17 cells are known to be regulated by specific transcription factors and cytokines, the role of microRNAs that control this inflammatory T cell subset and whether targeting microRNAs can have therapeutic effects are not understood well. We found that microRNA-21 expression is elevated in Th17 cells and Mir-21-/- mice show a defect in Th17 differentiation and are resistant to EAE. Mir-21-/- T cells fail to induce known factors required for Th17 differentiation, such as transcription factor RORgt and the cytokine IL-21. However, Mir-21-/- mice have normal Th1, Th2 and Treg differentiation. The T cell intrinsic function of Mir-21 was clearly demonstrated by the sensitivity to EAE of the Mir-21-/- mice into which WT CD4+ T cells were transferred. Mir-21 promoted Th17 differentiation by targeting Smad-7, a negative regulator of TGF-beta signaling and Th17 differentiation. Finally, we found that anti-Mir-21 treatment reduced clinical severity of EAE associated with decrease in Th17 cells. These findings demonstrate that Mir-21 confers susceptibility to EAE by affecting inflammatory Th17 responses and identify Mir-21 as a new target for therapeutic intervention in MS.

TLR4 Promotes Fibrosis but Attenuates Tubular Damage in Progressive Renal Injury

Toll-like receptors (TLRs) can orchestrate an inflammatory response upon activation by pathogen-associated motifs and release of endogenous stress ligands during tissue injury. The kidney constitutively expresses most TLRs, including TLR4. The function of TLR4 during the inflammation, tubular atrophy, and fibrosis that accompany progressive renal injury is unknown. Here, we subjected wild-type (WT) and TLR4-deficient mice to unilateral ureteral obstruction and observed elevated levels of TLR4 mRNA in the kidney after obstruction. One day after unilateral ureteral obstruction, TLR4-deficient mice had fewer proliferating tubular epithelial cells and more tubular damage than WT mice; however, TLR4-deficient mice developed considerably less renal fibrosis despite decreased matrix metalloproteinase activity and without significant differences in myofibroblast accumulation. In vitro, TLR4-deficient primary tubular epithelial cells and myofibroblasts produced significantly less type I collagen mRNA after TGF-beta stimulation than WT cells. The reduced fibrosis in TLR4-deficient mice associated with an upregulation of Bambi, a negative regulator of TGF-beta signaling. In conclusion, TLR4 attenuates tubular damage but promotes renal fibrosis by modulating the susceptibility of renal cells to TGF-beta. These data suggest that TLR4 signaling may be a therapeutic target for the prevention of renal fibrosis.

Tiul1 and TGIF are Involved in Downregulation of TGFβ1-induced IgA Isotype Expression

TGF-β1 is well known to induce Ig germ-line α (GLα) transcription and subsequent IgA isotype class switching recombination (CSR). Homeodomain protein TG-interacting factor (TGIF) and E3-ubiquitin ligases TGIF interacting ubiquitin ligase 1 (Tiul1) are implicated in the negative regulation of TGF-β signaling. In the present study, we investigated the roles of Tiul1 and TGIF in TGFβ1-induced IgA CSR. We found that over-expression of Tiul1 decreased TGFβ1-induced GLα promoter activity and strengthened the inhibitory effect of Smad7 on the promoter activity. Likewise, overexpression of TGIF also diminished GLα promoter activity and further strengthened the inhibitory effect of Tiul1, suggesting that Tiul1 and TGIF can down-regulate TGFβ1-induced GLα expression. In parallel, overexpression of Tiul1 decreased the expression of endogenous IgA CSR-predicitive transcripts (GLTα, PSTα, and CTα) and TGFβ1-induced IgA secretion, but not GLTγ3 and IgG3 secretion. Here, over-expressed TGIF further strengthened the inhibitory effect of Tiul1. These results suggest that Tiul1 and TGIF act as negatively regulators in TGFβ1-induced IgA isotype expression.

CO-Culture with Mesenchymal Cells Modulates TGF-Beta/Smad And Mapk Pathways in T Regulatory Cells

We previously demonstrated that when human bone marrow-derived mesenchymal cells (hMSCs) were co-cultured with CD4+/CD25+ T regulatory cells (Tregs) they maintained the T regulatory phenotype and function over time. Here we studied the TGF-beta/Smad signalling and mitogen-activated protein kinase (MAPK) pathways after Tregs/hMSCs co-culture. After 7 days co-culture of highly purified, immuno-selected Tregs and hMSCs from healthy donors, TGF-beta and IL-10 concentrations were dosed and mRNA of a panel of genes (NFAT, Smad4, Smad7, AP-1, Foxp3 and CD127) was quantified on harvested lymphocytes. Western blotting was performed by incubation with antiSMAD2, anti-pSMAD2, anti-ERK1/2 and anti-pERK1/2 polyclonal antibodies. After immuno-magnetic cell separation the final Treg fraction showed a mean purity of 93.6%±1. The CD4+/CD25+bright constituted 29%±7 of Tregs, FoxP3 cells constituted 51.9%±15.1 and CD127+ cells 19%±11.5. CD4+/CD25+ cells inhibited CD4+/CD25− cells (mean inhibition percentage: 52.1±29.6% (ratio 1:1). Tregs produced no TGF-beta and only a small quantity of IL-10 (8.67±4 pg/ml). hMSCs produced high quantity of TGF-beta (229.3 ± 54.8 pg/ml) associated with little IL-10 (2.7 ± 1.2 pg/ml). After co-culture, the TGF-beta concentration was 91.5±48.5 pg/ml while the IL-10 concentration was no different to baseline. To identify TGF-beta signaling pathways, we focused on Smad2, a central element in the cascade. In Tregs the strongly expressed pSmad2 signal after selection rapidly decreased after 7 days culture. When Tregs were co-cultured in presence of hMSCs the phosphorylated form of Smad2 did not change, indicating TGF-beta signaling was up-regulated. After co-culture mRNA quantification showed hMSCs down-regulated expression of SMAD7 (−8.9± 4.4 times vs Tregs without hMSCs), a negative regulator of TGF-beta signaling. After co-culture with hMSCs non-regulatory CD4+/CD25− control cells did not show any differences in SMAD7 expression. To study the MAPK kinase pathways, harvested Tregs were assayed for ERK1/2 kinase activity by determining their phosphorylated forms. pERK1/2 was almost absent in selected Tregs; it rapidly increased after 7 days in vitro culture without hMSCs but remained low after co-culture with hMSCs, indicating impaired ERK1/2 activation. AP-1 activation was also reduced (AP-1 mRNA −32±29 times less than controls). In conclusion, co-cultures of hMSCs and Tregs have the potential to set up a positive feedback loop that heightens responsiveness to TFG-beta in Treg cells. In fact, hMSCs produce TGF-beta which is consumed in co-culture with Tregs; TGF-beta1 signaling in Tregs is maintained through pSMAD2 up-regulation and SMAD7 down-regulation; hMSCs also inhibit ERK phosphorylation which consequently down-regulates AP-1 mRNA. Thus Tregs signalling is maintained through culture on a layer of hMSCs. Manipulation of Tregs signaling through culture on a layer of hMSCs may represent a novel strategy for maintenance of Treg phenotype and function.

Smad7 is inactivated through a direct physical interaction with the LIM protein Hic-5/ARA55

We recently reported that hydrogen peroxide-inducible clone-5 (Hic-5, also named androgen receptor-associated protein 55) can bind to the transforming growth factor-beta (TGF-beta)-signaling regulator Smad3, thereby inhibiting certain Smad3-dependent TGF-beta responses. We now show that Hic-5 can also control TGF-beta responses through an alternative mechanism involving Smad7, a key negative regulator of TGF-beta signaling. Hic-5 binds directly to Smad7. This interaction requires the LIM3 domain of Hic-5, and enhances TGF-beta signaling through causing loss of Smad7 protein but not mRNA. Enforced expression of Hic-5 reverses the ability of Smad7 to suppress TGF-beta-induced phosphorylation of Smads 2 and 3 and activation of the plasminogen activator inhibitor-1 promoter (in NRP-154 and PC3 prostate carcinoma and WPMY-1 prostate myofibroblast cell lines). Lentiviral-mediated small-hairpin RNA silencing of endogenous Hic-5 reduced TGF-beta responses in PC3 and WPMY-1 cells. Further work suggests that the level of Smad7 is modulated by its physical interaction with Hic-5 and targeted to a degradation pathway not likely to be proteasomal. Our findings support that Hic-5 functions as a cell-type-specific activator of TGF-beta signaling through its ability to physically interact with and neutralize Smad7.

Prognostic significance of BMP and activin membranebound inhibitor in colorectal cancer

To investigate the clinical significance of BMP and activin membrane-bound inhibitor (BAMBI) which is a pseudoreceptor of transforming growth factor-beta (TGF-beta) type I receptors and acts as a negative regulator of TGF-beta signaling and expression aberrantly elevated in colorectal cancers (CRCs). We studied BAMBI expression in CRCs. We studied BAMBI expression in 183 surgically resected CRCs by immunochemical and immunoblotting analyses using a generated monoclonal anti-BAMBI antibody. Commercially available anti-beta-catenin and anti-p53 antibodies were also applied for immunochemical analyses as a comparison control. Immunohistochemical analysis revealed that BAMBI expression was observed in 148 (80.8%), and strong BAMBI expression was observed in 46% of the CRCs. Strong BAMBI expression was positively correlated with histological type, depth of invasion, lymph node metastases, and tumor node metastasis (TNM) stage (P < 0.05). Clear associations were found between BAMBI and beta-catenin (P = 0.035) and p53 (P = 0.049) expression. In curatively resected CRC, 5-year recurrence-free survival was 51.9% (P = 0.037) for strong BAMBI expression compared to 79.8% for weak BAMBI expression. In the Cox's multivariate analysis, lymph node metastases (RR 6.685; P < 0.001) and depth of invasion (RR 14.0; P = 0.013) were significant indicators for recurrence, and strong BAMBI expression (RR 2.26; P = 0.057) tended to be significant. BAMBI was linked to a potentially aggressive tumor phenotype and predicted tumor recurrence and cancer-related death in CRC. BAMBI expression might be applicable in the routine clinical setting of CRC.

Involvement of the constitutive complex formation of c‐Ski/SnoN with smads in the impaired negative feedback regulation of transforming growth factor β signaling in scleroderma fibroblasts

The principal effect of transforming growth factor beta1 (TGFbeta1) on mesenchymal cells is its stimulation of extracellular matrix synthesis. Previous reports indicated the significance of the autocrine TGFbeta loop in the pathogenesis of scleroderma. The aim of this study was to examine c-Ski and SnoN, principal molecules in the negative regulation of TGFbeta signaling, to further understand the autocrine TGFbeta loop in scleroderma. Levels of expression of c-Ski/SnoN on cultured normal and scleroderma fibroblasts were determined by Western blotting, Northern blotting, and immunohistochemical staining. To determine the protein-protein interaction between c-Ski/SnoN, Smads, and p300, immunoprecipitation was performed. A transient transfection assay was performed to measure promoter activity of the alpha2(I) collagen gene and the 3TP-Lux plasmid construct. Scleroderma fibroblasts exhibited increased c-Ski/SnoN levels compared with normal fibroblasts, both in vivo and in vitro. Although c-Ski/SnoN constitutively formed a complex with Smads by immunoprecipitation, the inhibitory effect of c-Ski/SnoN on the promoter activity of human alpha2(I) collagen and 3TP-Lux was impaired in scleroderma fibroblasts. Immunoprecipitation analyses also revealed that overexpressed c-Ski/SnoN could not compete with p300 in these cells. These results indicate that impaired competition with p300 is the possible cause of dysfunction of c-Ski/SnoN in scleroderma fibroblasts and that this might contribute to maintenance of the autocrine TGFbeta loop in this disease.

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

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

TGF-β Regulation by Emilin1: New Links in the Etiology of Hypertension

Hypertension is a complex disease influenced by multiple genetic and environmental factors. The TGF-beta signaling pathway has a long recognized role in blood pressure homeostasis. In this issue of Cell, Zacchigna et al. (2006) report that the secreted protein Emilin1 is a negative regulator of TGF-beta signaling. Emilin1 knockout mice display elevated blood pressure due to increased TGF-beta signaling in the vasculature.

The novel E3 ubiquitin ligase Tiul1 associates with TGIF to target Smad2 for degradation.

Ubiquitin-dependent degradation plays an important role in the negative regulation of TGF-beta signaling. Here, we identify Tiul1 (for TGIF interacting ubiquitin ligase 1), a novel E3 ubiquitin ligase that inhibits TGF-beta signaling by targeting both the activated receptor and Smad2 for degradation. Tiul1 associates constitutively with Smad7 and induces degradation of the activated type I receptor without affecting the expression levels of Smad7. Tiul1 can also interact with Smad2 and the nuclear corepressor TGIF upon activation of TGF-beta signaling. Like Smad7, the steady-state levels of TGIF are not affected by Tiul1, but the interaction of Tiul1 with TGIF allows this ubiquitin ligase to target Smad2 for degradation. Consistent with this, overexpression of Tiul1 suppressed TGF-beta-induced growth arrest and transcriptional responses. In addition, silencing of Tiul1 or TGIF genes by siRNA resulted in suppression of the TGF-beta-dependent degradation of Smad2 and an enhancement of TGF-beta-mediated gene expression. These results reveal a new role for TGIF as a component of a ubiquitin ligase complex that mediates the degradation of Smad2 in response to TGF-beta signaling.

Impaired Smad7-Smurf-mediated negative regulation of TGF-beta signaling in scleroderma fibroblasts.

The principal effect of TGF-beta1 on mesenchymal cells is its stimulation of ECM synthesis. Previous reports indicated the significance of the autocrine TGF-beta loop in the pathogenesis of scleroderma. In this study, we focused on Smad7 and Smurfs, principal molecules in the negative regulation of TGF-beta signaling, to further understand the autocrine TGF-beta loop in scleroderma. Scleroderma fibroblasts exhibited increased Smad7 levels compared with normal fibroblasts in vivo and in vitro. Smad7 constitutively formed a complex with the TGF-beta receptors, and the inhibitory effect of Smad7 on the promoter activity of human alpha2(I) collagen and 3TP-lux was completely impaired in scleroderma fibroblasts. Furthermore, the protein stability of TGF-beta receptor type I was significantly increased in scleroderma fibroblasts compared with normal fibroblasts. There was no significant difference in Smurf1 and Smurf2 levels between normal and scleroderma fibroblasts, and the transiently overexpressed Smurf1 and/or Smurf2 did not affect TGF-beta receptor type I protein levels in scleroderma fibroblasts. These results indicate that the impaired Smad7-Smurf-mediated inhibitory effect on TGF-beta signaling might contribute to maintaining the autocrine TGF-beta loop in scleroderma fibroblasts. To our knowledge, this is the first report of a disturbed negative regulation of TGF-beta signaling in fibrotic disorders.

DACH1 Inhibits Transforming Growth Factor-β Signaling through Binding Smad4

The vertebrate homologues of Drosophila dachsund, DACH1 and DACH2, have been implicated as important regulatory genes in development. DACH1 plays a role in retinal and pituitary precursor cell proliferation and DACH2 plays a specific role in myogenesis. DACH proteins contain a domain (DS domain) that is conserved with the proto-oncogenes Ski and Sno. Since the Ski/Sno proto-oncogenes repress AP-1 and SMAD signaling, we hypothesized that DACH1 might play a similar cellular function. Herein, DACH1 was found to be expressed in breast cancer cell lines and to inhibit transforming growth factor-beta (TGF-beta)-induced apoptosis. DACH1 repressed TGF-beta induction of AP-1 and Smad signaling in gene reporter assays and repressed endogenous TGF-beta-responsive genes by microarray analyses. DACH1 bound to endogenous NCoR and Smad4 in cultured cells and DACH1 co-localized with NCoR in nuclear dotlike structures. NCoR enhanced DACH1 repression, and the repression of TGF-beta-induced AP-1 or Smad signaling by DACH1 required the DACH1 DS domain. The DS domain of DACH was sufficient for NCoR binding at a Smad4-binding site. Smad4 was required for DACH1 repression of Smad signaling. In Smad4 null HTB-134 cells, DACH1 inhibited the activation of SBE-4 reporter activity induced by Smad2 or Smad3 only in the presence of Smad4. DACH1 participates in the negative regulation of TGF-beta signaling by interacting with NCoR and Smad4.

Chromosomal Region Maintenance 1 (CRM1)-dependent Nuclear Export of Smad Ubiquitin Regulatory Factor 1 (Smurf1) Is Essential for Negative Regulation of Transforming Growth Factor-β Signaling by Smad7

Smad ubiquitin regulatory factor 1 (Smurf1), a HECT type E3 ubiquitin ligase, interacts with inhibitory Smad7 and induces translocation of Smad7 to the cytoplasm. Smurf1 then associates with the transforming growth factor (TGF)-beta type I receptor, TbetaR-I, enhancing turnover. However, the mechanism of nuclear export of Smad7 by Smurf1 has not been elucidated. Here we identified a functional nuclear export signal (NES) in a C-terminal region of Smurf1. In transfected cells, the Smurf1-Smad7 complex was accumulated in the cytoplasm by the nuclear export receptor, CRM1; this action was prevented by treatment with leptomycin B, a specific inactivator of CRM1 function. A green fluorescence protein fusion protein containing the C-terminal NES motif of Smurf1, located in the cytoplasm, accumulated in the nucleus following treatment with leptomycin B. Moreover, Smurf1 was shown to bind physically to CRM1 through NES, and nuclear export of the Smurf1-Smad7 complex was prevented by mutations of Smurf1 within the NES. Finally, the Smurf1 NES mutant reduced inhibition by Smad7 of the transcriptional activation induced by TGF-beta. These results thus suggest that CRM1-dependent nuclear export of Smurf1 is essential for the negative regulation of TGF-beta signaling by Smad7.