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.     

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.     

Concomitant targeting of EGF receptor, TGF-beta and SRC points to a novel therapeutic approach in pancreatic cancer

To test the hypothesis that concomitant targeting of the epidermal growth factor receptor (EGFR) and transforming growth factor-beta (TGF-β) may offer a novel therapeutic approach in pancreatic cancer, EGFR silencing by RNA interference (shEGFR) was combined with TGF-β sequestration by soluble TGF-β receptor II (sTβRII). Effects on colony formation in 3-dimensional culture, tumor formation in nude mice, and downstream signaling were monitored. In both ASPC-1 and T3M4 cells, either shEGFR or sTβRII significantly inhibited colony formation. However, in ASPC-1 cells, combining shEGFR with sTβRII reduced colony formation more efficiently than either approach alone, whereas in T3M4 cells, shEGFR-mediated inhibition of colony formation was reversed by sTβRII. Similarly, in vivo growth of ASPC-1-derived tumors was attenuated by either shEGFR or sTβRII, and was markedly suppressed by both vectors. By contrast, T3M4-derived tumors either failed to form or were very small when EGFR alone was silenced, and these effects were reversed by sTβRII due to increased cancer cell proliferation. The combination of shEGFR and sTβRII decreased phospho-HER2, phospho-HER3, phoshpo-ERK and phospho-src (Tyr416) levels in ASPC-1 cells but increased their levels in T3M4 cells. Moreover, inhibition of both EGFR and HER2 by lapatinib or of src by SSKI-606, PP2, or dasatinib, blocked the sTβRII-mediated antagonism of colony formation in T3M4 cells. Together, these observations suggest that concomitantly targeting EGFR, TGF-β, and src may constitute a novel therapeutic approach in PDAC that prevents deleterious cross-talk between EGFR family members and TGF-β-dependent pathways.     

髓性TGF-β受体Ⅱ敲除鼠的建立及其巨噬细胞表型的初步分析

目的:建立髓性TGF-β受体Ⅱ (TβRⅡ)敲除鼠模型并对其巨噬细胞表型进行了初步分析.方法:利用溶菌酶M启动子-重组酶转基因鼠(lysozyme M-Cre鼠)和TβRⅡ条件敲除鼠,建立定向髓性TβRⅡ敲除鼠,通过基因型检测、免疫细胞的分布组成,然后检测敲除鼠巨噬细胞细胞因子表达的变化及对肿瘤细胞凋亡的影响.结果:建立了髓性TβRⅡ敲除鼠,并初步证明,在肿瘤细胞上清刺激条件下,TβRⅡ敲除的巨噬细胞与对照细胞相比,CXCL1表达量下调.结论:TGF-β信号可调控巨噬细胞的CXCL1表达.     

TGF-β signalling is mediated by two autonomously functioning TβRI:TβRII pairs

Transforming growth factor (TGF)-βs are dimeric polypeptides that have vital roles in regulating cell growth and differentiation. They signal by assembling a receptor heterotetramer composed of two TβRI:TβRII heterodimers. To investigate whether the two heterodimers bind and signal autonomously, one of the TGF-β protomers was substituted to block receptor binding. The substituted dimer, TGF-β3 WD, bound the TβRII extracellular domain and recruited the TβRI with affinities indistinguishable from TGF-β3, but with one-half the stoichiometry. TGF-β3 WD was further shown to retain one-quarter to one-half the signalling activity of TGF-β3 in three established assays for TGF-β function. Single-molecule fluorescence imaging with GFP-tagged receptors demonstrated a measurable increase in the proportion of TβRI and TβRII dimers upon treatment with TGF-β3, but not with TGF-β3 WD. These results provide evidence that the two TβRI:TβRII heterodimers bind and signal in an autonomous manner. They further underscore how the TGF-βs diverged from the bone morphogenetic proteins, the ancestral ligands of the TGF-β superfamily that signal through a RI:RII:RII heterotrimer.     

Role of receptor complexes in resistance or sensitivity to growth inhibition by TGFβ in intestinal epithelial cell clones

Untransformed rat intestinal epithelial cells (IEC-18) were chemically mutagenized, selected in the presence of TGFβ₁, and cloned by limiting dilution. Two clones (4–5, 4–6) were resistant to growth inhibition by both TGFβ₁ and TGFβ₂. Another clone (4–1) was more sensitive to both TGFβ isoforms (relative to parental IEC-18 cells). IC₅₀ values for TGFβ_{1 and 2} in the 4–1 cells were at least 1/9 those of the parental cells; growth rates were reduced by 49% for TGFβ₁ and by 26% for TGFβ₂ in this clone. This increased sensitivity to TGFβ was explained by the 5- to 10-fold increase, relative to parental cells, in binding of TGFβ₁ and TGFβ₂ to both the type I and II receptors. In contrast, the resistance to growth inhibition by TGFβ in the 4–5 and 4–6 cells could not be explained by a decrease in either TGFβ binding affinities or in total number of receptors expressed, by the presence of serum binding components, or by occupation of receptor binding sites with autocrine TGF-β₁. However, in comparison to TGFβ-sensitive cells (IEC-18, 4–1), the resistant cells displayed a higher ratio of type II relative to type I receptor binding by TGF-β₁. Thus, a critical ratio of binding to receptor subtypes correlated with growth inhibition by TGF-β₁. Resistance to TGF-β₂ in the same clones did not appear to be receptor related. Thus different mechanisms for resistance to TGF-β₁ and TGF-β₂ were observed within a given clone. © 1993 Wiley-Liss, Inc.     

Cutting edge: in the absence of TGF-β signaling in T cells, fewer CD103+ regulatory T cells develop, but exuberant IFN-γ production renders mice more susceptible to helminth infection

Multiple factors control susceptibility of C57BL/6 mice to infection with the helminth Heligmosomoides polygyrus, including TGF-β signaling, which inhibits immunity in vivo. However, mice expressing a T cell-specific dominant-negative TGF-β receptor II (TGF-βRII DN) show dampened Th2 immunity and diminished resistance to infection. Interestingly, H. polygyrus-infected TGF-βRII DN mice show greater frequencies of CD4(+)Foxp3(+)Helios(+) Tregs than infected wild-type mice, but levels of CD103 are greatly reduced on both these cells and on the CD4(+)Foxp3(+)Helios(-) population. Although Th9 and Th17 levels are comparable between infected TGF-βRII DN and wild-type mice, the former develop exaggerated CD4(+) and CD8(+) T cell IFN-γ responses. Increased susceptibility conferred by TGF-βRII DN expression was lost in IFN-γ-deficient mice, although they remained unable to completely clear infection. Hence, overexpression of IFN-γ negatively modulates immunity, and the presence of Helios(+) Tregs may maintain susceptibility on the C57BL/6 background.     

Peptide ligands that use a novel binding site to target both TGF-β receptors

The transforming growth factor beta (TGF-β) signaling pathway plays myriad roles in development and disease. TGF-β isoforms initiate signaling by organizing their cell surface receptors TβRI and TβRII. Exploration and exploitation of the versatility of TGF-β signaling requires an enhanced understanding of structure-function relationships in this pathway. To this end, small molecule, peptide, and antibody effectors that bind key signaling components would serve as valuable probes. We focused on the extracellular domain of TβR1 (TβRI-ED) as a target for effector screening. The observation that TβRI-ED can bind to a TGF-β coreceptor (endoglin) suggests that the TβRI-ED may have multiple interaction sites. Using phage display, we identified two peptides LTGKNFPMFHRN (Pep1) and MHRMPSFLPTTL (Pep2) that bind the TβRI-ED (K(d)≈ 10(-5) M). Although our screen focused on TβRI-ED, the hit peptides interact with the TβRII-ED with similar affinities. The peptide ligands occupy the same binding sites on TβRI and TβRII, as demonstrated by their ability to compete with each other for receptor binding. Moreover, neither interferes with TGF-β binding. These results indicate that both TβRI and TβRII possess hot spots for protein-protein interactions that are distinct from those used by their known ligand TGF-β. To convert these compounds into high affinity probes, we exploited the observation that TβRI and TβRII exist as dimers on the cell surface; therefore, we assembled a multivalent ligand. Specifically, we displayed one of our receptor-binding peptides on a dendrimer scaffold. We anticipate that the potent multivalent ligand that resulted can be used to probe the role of receptor assembly in TGF-β function.     

Single-molecule imaging revealed enhanced dimerization of transforming growth factor β type II receptors in hypertrophic cardiomyocytes

Transforming growth factor β (TGF-β) signaling plays an important role in the pathogenesis of cardiac hypertrophy. However, the molecular mechanism of TGF-β signaling during the process of cardiac remodeling remains poorly understood. In the present study, by employing single-molecule fluorescence imaging approach, we demonstrated that in neonatal rat cardiomyocytes, TGF-β type II receptors (TβRII) existed as monomers at the low expression level, and dimerized upon TGF-β1 stimulation. Importantly, for the first time, we found the increased dimerization of TβRII in hypertrophic cardiomyocytes comparing to the normal cardiomyocytes. The enhanced TβRII dimerization was correlated with the enhanced Smad3 phosphorylation levels. These results provide new information on the mechanism of TGF-β signaling in cardiac remodeling.     

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.     

Transforming growth factor-β signaling: Tumorigenesis and targeting for cancer therapy

Transforming growth factor (TGF)-β is a multitasking cytokine such that its aberrant expression is related to cancer progression and metastasis. TGF-β is produced by a variety of cells within the tumor microenvironment (TME), and it is responsible for regulation of the activity of cells within this milieu. TGF-β is a main inducer of epithelial–mesenchymal transition (EMT), immune evasion, and metastasis during cancer progression. TGF-β exerts most of its functions by acting on TβRI and TβRII receptors in canonical (Smad-dependent) or noncanonical (Smad-independent) pathways. Members of mitogen-activated protein kinase, phosphatidylinositol 3-kinase/protein kinase B, and nuclear factor κβ are involved in the non-Smad TGF-β pathway. TGF-β acts by complex signaling, and deletion in one of the effectors in this pathway may influence the outcome in a diverse way by taking even an antitumor role. The stage and the type of tumor (contextual cues from cancer cells and/or the TME) and the concentration of TGF-β are other important factors determining the fate of cancer (progression or repression). There are a number of ways for targeting TGF-β signaling in cancer, among them the special focus is on TβRII suppression.     

Tamoxifen induces TGF-β1 activity and apoptosis of human MCF-7 breast cancer cells in vitro

We report here that the antiestrogen tamoxifen (TAM) induces cell death in human breast cancer cell line MCF-7. We assessed the type of cell death induced by TAM in this breast cancer cell line on the basis of morphological and biochemical characteristics. Dying cells showed morphological characteristics of apoptosis, such as chromatin condensation and nuclear disintegration. DNA isolated from these cells revealed a pattern of distinctive DNA bands on agarose gel. The DNA fragmentation in MCF-7 cells induced by TAM could also be detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling. Northern blot hybridization revealed a substantial increase in the amounts of TRPM-2 and TGF-β1 mRNAs in MCF-7 cells after treatment with TAM. In contrast, the mRNA level of the estrogen-induced pS2 gene was strongly suppressed. The biological activity of TGF-β was increased at least fourfold in the media from MCF-7 cells treated with TAM. The results presented in this study suggest that TAM induces apoptosis of MCF-7 cells and it may be mediated by the secretion of active TGF-β. © 1996 Wiley-Liss, Inc.     

The presence of Estrogen Receptor β modulates the response of breast cancer cells to therapeutic agents

Breast cancer is a leading cause of death for women. The estrogen receptors (ERs) ratio is important in the maintenance of mitochondrial redox status, and higher levels of ERβ increases mitochondrial functionality, decreasing ROS production. Our aim was to determine the interaction between the ERα/ERβ ratio and the response to cytotoxic treatments such as cisplatin (CDDP), paclitaxel (PTX) and tamoxifen (TAM). Cell viability, apoptosis, autophagy, ROS production, mitochondrial membrane potential, mitochondrial mass and mitochondrial functionality were analyzed in MCF-7 (high ERα/ERβ ratio) and T47D (low ERα/ERβ ratio) breast cancer cell lines. Cell viability decreased more in MCF-7 when treated with CDDP and PTX. Apoptosis was less activated after cytotoxic treatments in T47D than in MCF-7 cells. Nevertheless, autophagy was increased more in CDDP-treated MCF-7, but less in TAM-treated cells than in T47D. CDDP treatment produced a raise in mitochondrial mass in MCF-7, as well as the citochrome c oxidase (COX) and ATP synthase protein levels, however significantly reduced COX activity. In CDDP-treated cells, the overexpression of ERβ in MCF-7 caused a reduction in apoptosis, autophagy and ROS production, leading to higher cell survival; and the silencing of ERβ in T47D cells promoted the opposite effects. In TAM-treated cells, ERβ-overexpression led to less cell viability by an increment in autophagy; and the partial knockdown of ERβ in T47D triggered an increase in ROS production and apoptosis, leading to cell death. In conclusion, ERβ expression plays an important role in the response of cancer cells to cytotoxic agents, especially for cisplatin treatment.