TGF-β sensitivity is determined by N-linked glycosylation of the type II TGF-β receptor

N-linked glycosylation is a critical determinant of protein structure and function, regulating processes such as protein folding, stability and localization, ligand-receptor binding and intracellular signalling. TβRII [type II TGF-β (transforming growth factor β) receptor] plays a crucial role in the TGF-β signalling pathway. Although N-linked glycosylation of TβRII was first demonstrated over a decade ago, it was unclear how this modification influenced TβRII biology. In the present study, we show that inhibiting the N-linked glycosylation process successfully hinders binding of TGF-β1 to TβRII and subsequently renders cells resistant to TGF-β signalling. The lung cancer cell line A549, the gastric carcinoma cell line MKN1 and the immortal cell line HEK (human embryonic kidney)-293 exhibit reduced TGF-β signalling when either treated with two inhibitors, including tunicamycin (a potent N-linked glycosylation inhibitor) and kifunensine [an inhibitor of ER (endoplasmic reticulum) and Golgi mannosidase I family members], or introduced with a non-glycosylated mutant version of TβRII. We demonstrate that defective N-linked glycosylation prevents TβRII proteins from being transported to the cell surface. Moreover, we clearly show that not only the complex type, but also a high-mannose type, of TβRII can be localized on the cell surface. Collectively, these findings demonstrate that N-linked glycosylation is essentially required for the successful cell surface transportation of TβRII, suggesting a novel mechanism by which the TGF-β sensitivity can be regulated by N-linked glycosylation levels of TβRII.     

Roles for the type III TGF-β receptor in human cancer

Transforming growth factor β (TGF-β) superfamily ligands have important roles in regulating cellular homeostasis, embryonic development, differentiation, proliferation, immune surveillance, angiogenesis, motility, and apoptosis in a cell type and context specific manner. TGF-β superfamily signaling pathways also have diverse roles in human cancer, functioning to either suppress or promote cancer progression. The TGF-β superfamily co-receptor, the type III TGF-β receptor (TβRIII, also known as betaglycan) mediates TGF-β superfamily ligand dependent as well as ligand independent signaling to both Smad and non-Smad signaling pathways. Loss of TβRIII expression during cancer progression and direct effects of TβRIII on regulating cell migration, invasion, proliferation, and angiogenesis support a role for TβRIII as a suppressor of cancer progression and/or as a metastasis suppressor. Defining the physiological function and mechanism of TβRIII action and alterations in TβRIII function during cancer progression should enable more effective targeting of TβRIII and TβRIII mediated functions for the diagnosis and treatment of human cancer.     

Evaluation of the prognostic value of TGF-β superfamily type I receptor and TGF-β type II receptor expression in nasopharyngeal carcinoma using high-throughput tissue microarrays

Gene expression profiling had revealed that TGF-β superfamily type I receptor (also known as activin receptor-like kinase-1, ALK1) and TGFβR2 (TGF-β type II receptor) were down-regulated in nasopharyngeal carcinoma (NPC) (P < 0.05, respectively). However, no study with significantly large clinical samples to address the relevance of ALK1 and TGFβR2 in NPC progression or in patient outcomes has been reported. This study aims to assess the possible correlations of ALK1 and TGFβR2 expression with NPC progression and their potential prognostic predictive ability in NPC outcomes. ALK1 and TGFβR2 mRNA and protein levels were detected by qRT-PCR and NPC tissue microarray (TMA), which included 742 tissue cores. Both mRNA and protein levels of ALK1 and TGFβR2 were significantly lower in the cancer tissues compared with the non-cancerous tissues (P < 0.05). Epstein-Barr virus small RNA (EBER-1) hybridization signals in NPC showed significant associations with ALK1 and TGFβR2 proteins (P = 0.000 and 0.003, respectively). In the final logistic regression analysis model, the abnormal expression of ALK1 and TGFβR2 were found to be independent contributors to nasopharyngeal carcinogenesis (P = 0.000 and 0.000, respectively). A survival analysis revealed that ALK1 (Disease Free Survival (DFS): P = 0.002, Overall Survival (OS): P = 0.007) and TGFβR2 (DFS: P = 0.072, OS: P = 0.045) could predict the prognosis of NPC patients. The positive expression of ALK1 and TGFβR2 were independent risk factors for DFS and OS in multivariate analyses (DFS: P = 0.001 and 0.420, respectively; OS: P = 0.018 and 0.047, respectively). These results suggest that ALK1 and TGFβR2 may be useful prognostic biomarkers in NPC.     

Pentabromopseudilin: a myosin V inhibitor suppresses TGF-β activity by recruiting the type II TGF-β receptor to lysosomal degradation

Pentabromopseudilin (PBrP) is a marine antibiotic isolated from the marine bacteria Pseudomonas bromoutilis and Alteromonas luteoviolaceus. PBrP exhibits antimicrobial, anti-tumour, and phytotoxic activities. In mammalian cells, PBrP is known to act as a reversible and allosteric inhibitor of myosin Va (MyoVa). In this study, we report that PBrP is a potent inhibitor of transforming growth factor-β (TGF-β) activity. PBrP inhibits TGF-β-stimulated Smad2/3 phosphorylation, plasminogen activator inhibitor-1 (PAI-1) protein production and blocks TGF-β-induced epithelial–mesenchymal transition in epithelial cells. PBrP inhibits TGF-β signalling by reducing the cell-surface expression of type II TGF-β receptor (TβRII) and promotes receptor degradation. Gene silencing approaches suggest that MyoVa plays a crucial role in PBrP-induced TβRII turnover and the subsequent reduction of TGF-β signalling. Because, TGF-β signalling is crucial in the regulation of diverse pathophysiological processes such as tissue fibrosis and cancer development, PBrP should be further explored for its therapeutic role in treating fibrotic diseases and cancer.     

Distinctive mechanism for sustained TGF-β signaling and growth inhibition: MEK1 activation-dependent stabilization of type II TGF-β receptors

There are multiple mechanisms by which cells evade TGF-β-mediated growth inhibitory effects. In this report, we describe a novel mechanism by which cells become resistant to TGF-β-mediated growth suppression. Although having all the components of the TGF-β signaling pathway, different cell lines, RL, HaCaT, and BJAB, have different sensitivities toward TGF-β-induced growth suppression. The TGF-β resistance of RL, a B-cell lymphoma cell line, was due to ligand-induced downregulation of TGF-β receptor II (TβRII) and only transient TGF-β induced nuclear translocation of Smad2 and Smad3. With low-dose phorbol 12-myristate 13-acetate (PMA) or anti-IgM treatment, TGF-β sensitivity was restored by stabilizing TβRII expression and sustaining TGF-β signaling. The MEK inhibitor, U0126, blocked both PMA- and anti-IgM-induced upregulation of TβRII. In HaCaT and BJAB, two TGF-β-sensitive cell lines, which had higher basal levels of phospho-MEK and TβRII compared with RL, U0126 induced downregulation of TβRII and blocked subsequent TGF-β signaling. Similar results were also obtained with normal B cells, where MEK1 inhibitor downregulated TβRII and subsequent TGF-β signaling. Constitutively active MEK1, but not constitutively active ERK2, induced upregulation of TβRII. Furthermore, TβRII physically interacted with the constitutively active MEK1, but not with wild-type MEK1, indicating involvement of active MEK1 in stabilizing TβRII. Collectively, our data suggest a novel mechanism for MEK1 in regulating the sensitivity to TGF-β signaling by stabilizing TβRII.     

Controlling angiogenesis by two unique TGF-β type I receptor signaling pathways

Genetic studies in mice and humans have revealed a pivotal function for transforming growth factor-beta (TGF-β) in vascular development and maintenance of vascular homeostasis. Mice deficient for various TGF-β signaling components develop an embryonic lethality due to vascular defects. In patients, mutations in TGF-β receptors have been linked to vascular dysplasia like Hereditary Hemorrhagic Telangiectasia (HHT) and pulmonary arterial hypertension (PAH). Besides indirect effects by regulating the expression of angiogenic regulators, TGF-β also has potent direct effects on endothelial cell growth and migration, and we have proposed that TGF-β regulates the activation state of the endothelium via two opposing type I receptor/Smad pathways, activin receptor-like kinase (ALK)1 and ALK5. TGF-β is also critical for the differentiation of mural precursors into pericytes and smooth muscle cells. Furthermore, defective paracrine TGF-β signaling between endothelial and neighboring mural cells may be responsible for a leaky vessel phenotype that is characteristic of HHT. In this review, we discuss our current understanding of the TGF-β signaling pathway and its regulation of endothelial and vascular smooth muscle cell function.     

TGF-β receptor type II and fetuin in the developing sheep neocortex

Fetuin shows a characteristic pattern of distribution in the developing neocortex in many mammalian species. Its expression is confined to early-appearing cortical-plate and later subplate neurons. A short 19 amino-acid sequence of fetuin shows a degree of homology to an 18 amino-acid sequence of the TGF-β type II receptor (TβR-II) and in vitro fetuin binds to members of the TGF-β family of cytokines. It has been suggested that fetuin is the biologically significant antagonist of these cytokines. We have compared, using immunocytochemistry, the distribution pattern of TβR-II and fetuin in the developing neocortex of foetal sheep. TβR-II immunoreactivity first appears at around 40 days of gestation in the fetal sheep (E40, term in sheep is 150 days from conception), localised in two discreet bands: one just outside the cortical plate in the inner part of the marginal zone and one deep in the cortical plate in what becomes the transient subplate zone. By E70–E80, TβR-II is prominent in a population of subplate cells, whereas, by E120 only small patches of TβR-II-positive cells are visible, principally in pyramidal cells in layer VI. The developmental sequence of the staining pattern for TβR-II in the neocortex is complementary to that for fetuin, rather than overlapping with it. Double-labelling of fetuin and TβR-II shows some cellular co-localisation, especially at E60, but most fetuin-positive cells are not immunoreactive for TβR-II. Thus, fetuin’s proposed role as an antagonist of TGF-β cytokines and mimic of TβR-II is not consistent with the observed distribution of these two molecules in the developing neocortex of the foetal sheep. Received: 20 March 1997 / Accepted: 12 May 1997     

TRAF6 mediates IL-1β/LPS-induced suppression of TGF-β signaling through its interaction with the type III TGF-β receptor

Transforming growth factor-β1 (TGF-β1) is an important anti-inflammatory cytokine that modulates and resolves inflammatory responses. Recent studies have demonstrated that inflammation enhances neoplastic risk and potentiates tumor progression. In the evolution of cancer, pro-inflammatory cytokines such as IL-1β must overcome the anti-inflammatory effects of TGF-β to boost pro-inflammatory responses in epithelial cells. Here we show that IL-1β or Lipopolysaccharide (LPS) suppresses TGF-β-induced anti-inflammatory signaling in a NF-κB-independent manner. TRAF6, a key molecule in IL-1β signaling, mediates this suppressive effect through interaction with the type III TGF-β receptor (TβRIII), which is TGF-β-dependent and requires type I TGF-β receptor (TβRI) kinase activity. TβRI phosphorylates TβRIII at residue S829, which promotes the TRAF6/TβRIII interaction and consequent sequestration of TβRIII from the TβRII/TβRI complex. Our data indicate that IL-1β enhances the pro-inflammatory response by suppressing TGF-β signaling through TRAF6-mediated sequestration of TβRIII, which may be an important contributor to the early stages of tumor progression.     

Small molecule-mediated TGF-β type II receptor degradation promotes cardiomyogenesis in embryonic stem cells

The cellular signals controlling the formation of cardiomyocytes, vascular smooth muscle, and endothelial cells from stem cell-derived mesoderm are poorly understood. To identify these signals, a mouse embryonic stem cell (ESC)-based differentiation assay was screened against a small molecule library resulting in a 1,4-dihydropyridine inducer of type II TGF-β receptor (TGFBR2) degradation-1 (ITD-1). ITD analogs enhanced proteasomal degradation of TGFBR2, effectively clearing the receptor from the cell surface and selectively inhibiting intracellular signaling (IC(50) ~0.4-0.8 μM). ITD-1 was used to evaluate TGF-β involvement in mesoderm formation and cardiopoietic differentiation, which occur sequentially during early development, revealing an essential role in both processes in ESC cultures. ITD-1 selectively enhanced the differentiation of uncommitted mesoderm to cardiomyocytes, but not to vascular smooth muscle and endothelial cells. ITD-1 is a highly selective TGF-β inhibitor and reveals an unexpected role for TGF-β signaling in controlling cardiomyocyte differentiation from multipotent cardiovascular precursors.     

Regulation of TGF-β receptor activity

Background

Thyroid hormone (T3) is important for adult organ function and vertebrate development. Amphibian metamorphosis is totally dependent on T3 and offers a unique opportunity to study how T3 controls postembryonic development in vertebrates. Earlier studies have demonstrated that TR mediates the metamorphic effects of T3 in Xenopus laevis. Liganded TR recruits histone modifying coactivator complexes to target genes during metamorphosis. This leads to nucleosomal removal and histone modifications, including methylation of histone H3 lysine (K) 79, in the promoter regions, and the activation of T3-inducible genes.

Results

We show that Dot1L, the only histone methyltransferase capable of methylating H3K79, is directly regulated by TR via binding to a T3 response element in the promoter region during metamorphosis in Xenopus tropicalis, a highly related species of Xenopus laevis. We further show that Dot1L expression in both the intestine and tail correlates with the transformation of the organs.

Conclusions

Our findings suggest that TR activates Dot1L, which in turn participates in metamorphosis through a positive feedback to enhance H3K79 methylation and gene activation by liganded TR.     

Screening and identification of a novel class of TGF-β type 1 receptor kinase inhibitor

Transforming growth factor β (TGF-β) type I receptor (activin receptor-like kinase 5, ALK5) has been identified as a promising target for fibrotic diseases. To find a novel inhibitor of ALK5, the authors performed a high-throughput screen of a library of 420,000 compounds using dephosphorylated ALK5. From primary hits of 1521 compounds, 555 compounds were confirmed. In total, 124 compounds were then selected for follow-up based on their unique structures and other properties. Repeated concentration-response testing and final interference assays of the above compounds resulted in the discovery of a structurally novel ALK5 inhibitor (compound 8) (N-(thiophen 2-ylmethyl)-3-(3,4,5 trimethoxyphenyl)imidazo[1,2β]pyridazin 6-amine) with a low IC(50) value of 0.7 μM. Compound 8 also inhibited the TGF-β-induced nuclear translocation of SMAD with an EC(50) value of 0.8 μM. Kinetic analysis revealed that compound 8 inhibited ALK5 via mixed-type inhibition, suggesting that it may bind to ALK5 differently than other published adenosine triphosphate site inhibitors.     

The TGF-β co-receptor, CD109, promotes internalization and degradation of TGF-β receptors

Transforming growth factor-β (TGF-β) is implicated in numerous pathological disorders, including cancer and mediates a broad range of biological responses by signaling through the type I and II TGF-β receptors. Internalization of these receptors via the clathrin-coated pits pathway facilitates SMAD-mediated signaling, whereas internalization via the caveolae pathway is associated with receptor degradation. Thus, molecules that modulate receptor endocytosis are likely to play a critical role in regulating TGF-β action. We previously identified CD109, a GPI-anchored protein, as a TGF-β co-receptor and a negative regulator of TGF-β signaling. Here, we demonstrate that CD109 associates with caveolin-1, a major component of the caveolae. Moreover, CD109 increases binding of TGF-β to its receptors and enhances their internalization via the caveolae. In addition, CD109 promotes localization of the TGF-β receptors into the caveolar compartment in the presence of ligand and facilitates TGF-β-receptor degradation. Thus, CD109 regulates TGF-β receptor endocytosis and degradation to inhibit TGF-β signaling.     

Retromer maintains basolateral distribution of the type II TGF-β receptor via the recycling endosome

Transforming growth factor β (TGF-β) is critical for the development and maintenance of epithelial structures. Because receptor localization and trafficking affect the cellular and organismal response to TGF-β, the present study was designed to address how such homeostatic control is regulated. To that end, we identify a new role for the mammalian retromer complex in maintaining basolateral plasma membrane expression of the type II TGF-β receptor (TβRII). Retromer and TβRII associate in the presence or absence of TGF-β ligand. After retromer knockdown, although TβRII internalization and trafficking to a Rab5-positive compartment occur as in wild-type cells, receptor recycling is inhibited. This results in TβRII mislocalization from the basolateral to both the basolateral and apical plasma membranes independent of Golgi transit and the Rab11-positive apical recycling endosome. The data support a model in which, after initial basolateral TβRII delivery, steady-state polarized TβRII expression is maintained by retromer/TβRII binding and delivery to the common recycling endosome.     

USP4 is regulated by AKT phosphorylation and directly deubiquitylates TGF-β type I receptor

The stability and membrane localization of the transforming growth factor-β (TGF-β) type I receptor (TβRI) determines the levels of TGF-β signalling. TβRI is targeted for ubiquitylation-mediated degradation by the SMAD7-SMURF2 complex. Here we performed a genome-wide gain-of-function screen and identified ubiquitin-specific protease (USP) 4 as a strong inducer of TGF-β signalling. USP4 was found to directly interact with TβRI and act as a deubiquitylating enzyme, thereby controlling TβRI levels at the plasma membrane. Depletion of USP4 mitigates TGF-β-induced epithelial to mesenchymal transition and metastasis. Importantly, AKT (also known as protein kinase B), which has been associated with poor prognosis in breast cancer, directly associates with and phosphorylates USP4. AKT-mediated phosphorylation relocates nuclear USP4 to the cytoplasm and membrane and is required for maintaining its protein stability. Moreover, AKT-induced breast cancer cell migration was inhibited by USP4 depletion and TβRI kinase inhibition. Our results uncover USP4 as an important determinant for crosstalk between TGF-β and AKT signalling?pathways.