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 signaling plays an essential role in the growth and maintenance of intervertebral disc tissue

TGF-β signaling plays a critical role in cartilage and spine tissue development at embryonic stage but its role in postnatal intervertebral disc (IVD) tissue growth and maintenance remain poorly understood. In the present studies, we have deleted the Tgfbr2 gene in inner annulus fibrosus cells of the disc tissue and surrounding growth plate chondrocytes using Col2a1-CreER(T2) transgenic mice. We found that TGF-β signaling is required for normal growth plate cartilage and endplate cartilage growth at postnatal stage. The expression of Mmp13 gene is significantly up-regulated in primary disc cells of Tgfbr2 conditional knockout mice. Deletion of the Mmp13 gene under Tgfbr2 null background completely reverses the abnormal disc phenotype found in Tgfbr2 knockout mice.     

TGF-β receptors: In and beyond TGF-β signaling

Transforming growth factor β (TGF-β) plays an important role in normal development and homeostasis. Dysregulation of TGF-β responsiveness and its downstream signaling pathways contribute to many diseases, including cancer initiation, progression, and metastasis. TGF-β ligands bind to three isoforms of the TGF-β receptor (TGFBR) with different affinities. TGFBR1 and 2 are both serine/threonine and tyrosine kinases, but TGFBR3 does not have any kinase activity. They are necessary for activating canonical or noncanonical signaling pathways, as well as for regulating the activation of other signaling pathways. Another prominent feature of TGF-β signaling is its context-dependent effects, temporally and spatially. The diverse effects and context dependency are either achieved by fine-tuning the downstream components or by regulating the expressions and activities of the ligands or receptors. Focusing on the receptors in events in and beyond TGF-β signaling, we review the membrane trafficking of TGFBRs, the kinase activity of TGFBR1 and 2, the direct interactions between TGFBR2 and other receptors, and the novel roles of TGFBR3.     

Crosstalk between TGF-β and hedgehog signaling in cancer

Hedgehog (HH) and TGF-β signals control various aspects of embryonic development and cancer progression. While their canonical signal transduction cascades have been well characterized, there is increasing evidence that these pathways are able to exert overlapping activities that challenge efficient therapeutic targeting. We herein review the current knowledge on HH signaling and summarize the recent findings on the crosstalks between the HH and TGF-β pathways in cancer.     

TGF-β and BMP signaling in osteoblast differentiation and bone formation

Transforming growth factor-beta (TGF-β)/bone morphogenic protein (BMP) signaling is involved in a vast majority of cellular processes and is fundamentally important throughout life. TGF-β/BMPs have widely recognized roles in bone formation during mammalian development and exhibit versatile regulatory functions in the body. Signaling transduction by TGF-β/BMPs is specifically through both canonical Smad-dependent pathways (TGF-β/BMP ligands, receptors and Smads) and non-canonical Smad-independent signaling pathway (e.g. p38 mitogen-activated protein kinase pathway, MAPK). Following TGF-β/BMP induction, both the Smad and p38 MAPK pathways converge at the Runx2 gene to control mesenchymal precursor cell differentiation. The coordinated activity of Runx2 and TGF-β/BMP-activated Smads is critical for formation of the skeleton. Recent advances in molecular and genetic studies using gene targeting in mice enable a better understanding of TGF-β/BMP signaling in bone and in the signaling networks underlying osteoblast differentiation and bone formation. This review summarizes the recent advances in our understanding of TGF-β/BMP signaling in bone from studies of genetic mouse models and human diseases caused by the disruption of TGF-β/BMP signaling. This review also highlights the different modes of cross-talk between TGF-β/BMP signaling and the signaling pathways of MAPK, Wnt, Hedgehog, Notch, and FGF in osteoblast differentiation and bone formation.     

Curcumin suppresses TGF-β signaling by inhibition of TGIF degradation in scleroderma fibroblasts

The transforming growth factor-β (TGF-β) signaling pathway plays a key role in the fibrotic process in systemic scleroderma (SSc). Curcumin, a Turmeric root extract, has been demonstrated to exert antifibrotic activity. In the present study, we carefully investigated the effect of curcumin on TGF-β signaling and its potential mechanism in SSc fibroblasts. We demonstrated a potent inhibitory effect of curcumin on TGF-β signaling. Curcumin counteracted TGF-β-induced phosphorylation of Smad2 but not Smad3. Further study revealed curcumin induced upregulation of TGF-β-induced factor (TGIF), a negative regulator of TGF-β signaling. The TGIF silencing results evidenced the essential role of TGIF in curcumin-mediated TGF-β/Smad2 suppression. Moreover, our data indicated that the upregulation of TGIF by curcumin might result from decreased ubiquitination of TGIF, which blocks its proteasome-mediated degradation. Collectively, our data provide a novel mechanism of curcumin-mediated suppression of fibrotic process in scleroderma.     

Ancient and diverged TGF-β signaling components in Nasonia vitripennis

The transforming growth factor beta (TGF)-β signaling pathway and its modulators are involved in many aspects of cellular growth and differentiation in all metazoa. Although most of the core components of the pathway are highly conserved, many lineage-specific adaptations have been observed including changes regarding paralog number, presence and absence of modulators, and functional relevance for particular processes. In the parasitic jewel wasp Nasonia vitripennis, the bone morphogenetic proteins (BMPs), one of the major subgroups of the TGF-β superfamily, play a more fundamental role in dorsoventral (DV) patterning than in all other insects studied so far. However, Nasonia lacks the BMP antagonist Short gastrulation (Sog)/chordin, which is essential for polarizing the BMP gradient along the DV axis in most bilaterian animals. Here, we present a broad survey of TGF-β signaling in Nasonia with the aim to detect other lineage-specific peculiarities and to identify potential mechanisms, which explain how BMP-dependent DV pattering occurs in the early Nasonia embryo in the absence of Sog.     

USP15 stabilizes TGF-β receptor I and promotes oncogenesis through the activation of TGF-β signaling in glioblastoma

In advanced cancer, including glioblastoma, the transforming growth factor β (TGF-β) pathway acts as an oncogenic factor and is considered to be a therapeutic target. Using a functional RNAi screen, we identified the deubiquitinating enzyme ubiquitin-specific peptidase 15 (USP15) as a key component of the TGF-β signaling pathway. USP15 binds to the SMAD7-SMAD specific E3 ubiquitin protein ligase 2 (SMURF2) complex and deubiquitinates and stabilizes type I TGF-β receptor (TβR-I), leading to an enhanced TGF-β signal. High expression of USP15 correlates with high TGF-β activity, and the USP15 gene is found amplified in glioblastoma, breast and ovarian cancer. USP15 amplification confers poor prognosis in individuals with glioblastoma. Downregulation or inhibition of USP15 in a patient-derived orthotopic mouse model of glioblastoma decreases TGF-β activity. Moreover, depletion of USP15 decreases the oncogenic capacity of patient-derived glioma-initiating cells due to the repression of TGF-β signaling. Our results show that USP15 regulates the TGF-β pathway and is a key factor in glioblastoma pathogenesis.     

TGF-β family signaling in stem cells

Background

The diversity of cell types and tissue types that originate throughout development derives from the differentiation potential of embryonic stem cells and somatic stem cells. While the former are pluripotent, and thus can give rise to a full differentiation spectrum, the latter have limited differentiation potential but drive tissue remodeling. Additionally cancer tissues also have a small population of self-renewing cells with stem cell properties. These cancer stem cells may arise through dedifferentiation from non-stem cells in cancer tissues, illustrating their plasticity, and may greatly contribute to the resistance of cancers to chemotherapies.

Scope of review

The capacity of the different types of stem cells for self-renewal, the establishment and maintenance of their differentiation potential, and the selection of differentiation programs are greatly defined by the interplay of signaling molecules provided by both the stem cells themselves, and their microenvironment, the niche. Here we discuss common and divergent roles of TGF-β family signaling in the regulation of embryonic, reprogrammed pluripotent, somatic, and cancer stem cells.

Major conclusions

Increasing evidence highlights the similarities between responses of normal and cancer stem cells to signaling molecules, provided or activated by their microenvironment. While TGF-β family signaling regulates stemness of normal and cancer stem cells, its effects are diverse and depend on the cell types and physiological state of the cells.

General significance

Further mechanistic studies will provide a better understanding of the roles of TGF-β family signaling in the regulation of stem cells. These basic studies may lead to the development of a new therapeutic or prognostic strategies for the treatment of cancers. This article is part of a Special Issue entitled Biochemistry of Stem Cells.     

Inhibiting TGF-β signaling in hepatocellular carcinoma

One of the main complications in patients with liver fibrosis is the development of hepatocellular carcinoma (HCC). An understanding of the molecular mechanisms leading to HCC is important in order to be able to design new pharmacological agents serving either to prevent or mitigate the outcome of this malignancy. The transforming growth factor-beta (TGF-β) cytokine and its isoforms initiate a signaling cascade which is closely linked to liver fibrosis, cirrhosis and subsequent progression to HCC. Because of its role in these stages of disease progression, TGF-β appears to play a unique role in the molecular pathogenesis of HCC. Thus, it is a promising target for pharmacological treatment strategies. Recent studies have shown that inhibition of TGF-β signaling results in multiple synergistic down-stream effects which will likely improve the clinical outcome in HCC. We also review a number of TGF-β inhibitors, most of which are still in a preclinical stage of development, but may soon be available for trial in HCC patients. Hence, it is anticipated that there will soon be new agents available for clinical investigations to evaluate the role of the TGF-β-associated signaling in this deadly cancer.     

Convergent signaling in the regulation of connective tissue growth factor in malignant mesothelioma: TGFβ signaling and defects in the Hippo signaling cascade

Malignant mesothelioma (MM) is a neoplasm that arises from serosal surfaces of the pleural, peritoneal and pericardial cavities with worldwide incidence, much of which is caused by asbestos exposure. Patients suffer from pain and dyspnea due to direct invasion of the chest wall, lungs and vertebral or intercostal nerves by masses of thick fibrotic tumors. Although there has been recent progress in the clinical treatment, current therapeutic approaches do not provide satisfactory results. Therefore, development of a molecularly targeted therapy for MM is urgently required. Our recent studies suggest that normal mesothelial and MM cell growth is promoted by TGFβ, and that TGFβ signaling together with intrinsic disturbances in neurofibromatosis type 2 (NF2) and Hippo signaling cascades in MM cells converges upon further expression of connective tissue growth factor (CTGF). The formation of a YAP-TEAD4–Smad3-p300 complex on the specific CTGF promoter site with an adjacent TEAD and Smad binding motif is a critical and synergistic event caused by the dysregulation of these two distinct cascades. Furthermore, we demonstrated the functional importance of CTGF through the mouse studies and human histological analyses, which may elucidate the clinical features of MM with severe fibrosis in the thoracic cavity.     

Convergent signaling in the regulation of connective tissue growth factor in malignant mesothelioma: TGFβ signaling and defects in the Hippo signaling cascade

Malignant mesothelioma (MM) is a neoplasm that arises from serosal surfaces of the pleural, peritoneal and pericardial cavities with worldwide incidence, much of which is caused by asbestos exposure. Patients suffer from pain and dyspnea due to direct invasion of the chest wall, lungs and vertebral or intercostal nerves by masses of thick fibrotic tumors. Although there has been recent progress in the clinical treatment, current therapeutic approaches do not provide satisfactory results. Therefore, development of a molecularly targeted therapy for MM is urgently required. Our recent studies suggest that normal mesothelial and MM cell growth is promoted by TGFβ, and that TGFβ signaling together with intrinsic disturbances in neurofibromatosis type 2 (NF2) and Hippo signaling cascades in MM cells converges upon further expression of connective tissue growth factor (CTGF). The formation of a YAP-TEAD4–Smad3-p300 complex on the specific CTGF promoter site with an adjacent TEAD and Smad binding motif is a critical and synergistic event caused by the dysregulation of these two distinct cascades. Furthermore, we demonstrated the functional importance of CTGF through the mouse studies and human histological analyses, which may elucidate the clinical features of MM with severe fibrosis in the thoracic cavity.     

Role of glycosylation in TGF-β signaling and epithelial-to-mesenchymal transition in cancer

Glycosylation is a common posttranslational modification on membrane-associated and secreted proteins that is of pivotal importance for regulating cell functions.Aberrant glycosylation can lead to uncontrolled cell proliferation,cell-matrix interactions,migration and differentiation,and has been shown to be involved in cancer and other diseases.The epithelial-to-mesenchymal transition is a key step in the metastatic process by which cancer cells gain the ability to invade tissues and extravasate into the bloodstream.This cellular transformation process,which is associated by morphological change,loss of epithelial traits and gain of mesenchymal markers,is triggered by the secreted cytokine transforming growth factor-β(TGF-β).TGF-βbioactivity is carefully regulated,and its effects on cells are mediated by its receptors on the cell surface.In this review,we first provide a brief overview of major types of glycans,namely,N-glycans,O-glycans,glycosphingolipids and glycosaminoglycans that are involved in cancer progression.Thereafter,we summarize studies on how the glycosylation of TGF-βsignaling components regulates TGF-βsecretion,bioavailability and TGF-βreceptor function.Then,we review glycosylation changes associated with TGF-β-induced epithelial-to-mesenchymal transition in cancer.Identifying and understanding the mechanisms by which glycosylation affects TGF-βsignaling and downstream biological responses will facilitate the identification of glycans as biomarkers and enable novel therapeutic approaches.     

Role of TGF-β signaling in the mechanisms of tamoxifen resistance

The transforming growth factor beta (TGF-β) signaling pathway plays complex role in the regulation of cell proliferation, apoptosis and differentiation in breast cancer. TGF-β activation can lead to multiple cellular responses mediating the drug resistance evolution, including the resistance to antiestrogens. Tamoxifen is the most commonly prescribed antiestrogen that functionally involved in regulation of TGF-β activity. In this review, we focus on the role of TGF-β signaling in the mechanisms of tamoxifen resistance, including its interaction with estrogen receptors alfa (ERα) pathway and breast cancer stem cells (BCSCs). We summarize the current reported data regarding TGF-β signaling components as markers of tamoxifen resistance and review current approaches to overcoming tamoxifen resistance based on studies of TGF-β signaling.     

Aging,osteoarthritis and transforming growth factor-β signaling in cartilage

Osteoarthritis is a common malady of the musculoskeletal system affecting the articular cartilage. The increased frequency of osteoarthritis with aging indicates the complex etiology of this disease, which includes pathophysiology and joint stability including biomechanics. The balance between anabolic morphogens and growth factors and catabolic cytokines is at the crux of the problem of osteoarthritis. One such signal is transforming growth factor-β (TGF-β). The impaired TGF-β signaling has been identified as a culprit in old mice in a recent article in this journal. This commentary places this discovery in the context of anabolic and catabolic signals and articular cartilage homeostasis in the joint.     

EWI-2 negatively regulates TGF-β signaling leading to altered melanoma growth and metastasis

In normal melanocytes, TGF-β signaling has a cytostatic effect. However, in primary melanoma cells, TGF-β-induced cytostasis is diminished, thus allowing melanoma growth. Later, a second phase of TGF-β signaling supports melanoma EMT-like changes, invasion and metastasis. In parallel with these “present-absent-present” TGF-β signaling phases, cell surface protein EWI motif-containing protein 2 (EWI-2 or IgSF8) is “absent-present-absent” in melanocytes, primary melanoma, and metastatic melanoma, respectively, suggesting that EWI-2 may serve as a negative regulator of TGF-β signaling. Using melanoma cell lines and melanoma short-term cultures, we performed RNAi and overexpression experiments and found that EWI-2 negatively regulates TGF-β signaling and its downstream events including cytostasis (in vitro and in vivo), EMT-like changes, cell migration, CD271-dependent invasion, and lung metastasis (in vivo). When EWI-2 is present, it associates with cell surface tetraspanin proteins CD9 and CD81 — molecules not previously linked to TGF-β signaling. Indeed, when associated with EWI-2, CD9 and CD81 are sequestered and have no impact on TβR2-TβR1 association or TGF-β signaling. However, when EWI-2 is knocked down, CD9 and CD81 become available to provide critical support for TβR2-TβR1 association, thus markedly elevating TGF-β signaling. Consequently, all of those TGF-β-dependent functions specifically arising due to EWI-2 depletion are reversed by blocking or depleting cell surface tetraspanin proteins CD9 or CD81. These results provide new insights into regulation of TGF-β signaling in melanoma, uncover new roles for tetraspanins CD9 and CD81, and strongly suggest that EWI-2 could serve as a favorable prognosis indicator for melanoma patients.