Inactivation of FAM20B causes cell fate changes in annulus fibrosus of mouse intervertebral disc and disc defects via the alterations of TGF-β and MAPK signaling pathways

Intervertebral disc (IVD) disorder is often caused by the defect of annulus fibrosus (AF), especially that of the outer AF. Studies about the mechanisms governing the development of the outer AF are needed for a better understanding of pathogenesis of IVD defects. Glycosaminoglycans (GAGs) are essential components of extracellular matrix (ECM) in AF. FAM20B is a newly identified xylose kinase that catalyzes the biosynthesis of GAGs. In this study, we created Fam20B conditional knockout (cKO) mice in which FAM20B was inactivated in type I collagen-expressing cells, the main type of cells in the outer AF of IVD. The cKO mice showed severe spine deformity and remarkable IVD defects associated with AF malformation. The AF of cKO mice had a lower level of chondroitin sulfate and heparan sulfate, and the outer AF cells lost their normal fibroblast-like morphology and acquired chondrocyte phenotypes, expressing a higher level of Sox 9 and type II collagen along with a reduced level of type I collagen. The level of phospho-Smad 2 and phospho-Smad 3, and that of scleraxis, a downstream target molecule of canonical TGF-β signaling pathway were significantly lower in the AF of cKO mice. The AF in cKO mice also manifested altered levels in the molecules associated with the activations of MAPK pathway; the changes included the increase of phospho-P38 and phospho-ERK and a decrease of phospho-JNK. These results indicate that FAM20B plays an essential role in the development of AF by regulating the TGF-β signaling and MAPK signaling pathways.     

Dietary fiber enhances TGF-β signaling and growth inhibition in the gut

Dietary fiber intake links to decreased risk of colorectal cancers. The underlying mechanisms remain unclear. Recently, we found that butyrate, a short-chain fatty acid produced in gut by bacterial fermentation of dietary fiber, enhances TGF-β signaling in rat intestinal epithelial cells (RIE-1). Furthermore, TGF-β represses inhibitors of differentiation (Ids), leading to apoptosis. We hypothesized that dietary fiber enhances TGF-β's growth inhibitory effects on gut epithelium via inhibition of Id2. In this study, Balb/c and DBA/2N mice were fed with a regular rodent chow or supplemented with a dietary fiber (20% pectin) and Smad3 level in gut epithelium was measured. In vitro, RIE-1 cells were treated with butyrate and TGF-β(1), and cell functions were evaluated. Furthermore, the role of Ids in butyrate- and TGF-β-induced growth inhibition was investigated. We found that pectin feeding increased Smad3 protein levels in the jejunum (1.47 ± 0.26-fold, P = 0.045, in Balb/c mice; 1.49 ± 0.19-fold, P = 0.016, in DBA/2N mice), and phospho-Smad3 levels (1.92 ± 0.27-fold, P = 0.009, in Balb/c mice; 1.83 ± 0.28-fold, P = 0.022, in DBA/2N mice). Butyrate or TGF-β alone inhibited cell growth and induced cell cycle arrest. The combined treatment of butyrate and TGF-β synergistically induced cell cycle arrest and apoptosis in RIE-1 cells and repressed Id2 and Id3 levels. Furthermore, knockdown of Id2 gene expression by use of small interfering RNA caused cell cycle arrest and apoptosis. We conclude that dietary fiber pectin enhanced Smad3 expression and activation in the gut. Butyrate and TGF-β induced cell cycle arrest and apoptosis, which may be mediated by repression of Id2. Our results implicate a novel mechanism of dietary fiber in reducing the risk of colorectal cancer development.     

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.     

Effects of bio-active ceramic resources in cutaneous wound healing and the role of TGF-β signaling

The wound healing process is a highly orchestrated process, which includes inflammation, re-epithelialization, granulation tissue formation, matrix formation and re-modeling. In this paper, we attempt to determine if bio-active ceramic resource powder particles had an effect on cutaneous wound healing. Furthermore, we investigated its related mechanism and the expression of Smads of cutaneous wound healing, which can be accelerated by bio-active ceramic ointment. Topically applied lesions of 5%, 10% and 15% bio-active ceramic ointment (AO) showed accelerated wound closure, re-epithelialization, and the related immediate down stream of TGF-β (p-Smad2/3 and Smad3) was suppressed. In particular, 10% and 15% AO lesions became closed faster at Days 3 and 4 of post-wound and p-Smad2/3 was also suppressed. All AO lesions showed accelerated mild wound closure at Day 6, but there were no significant difference. Several papers reported that Smad3 may mediate the signaling pathways that is inhibitory to wound healing, as the deletion of Smad3 leads to enhanced re-epithelialization and contraction of the wound area. This study showed that topical, bio-active ceramic ointment applications accelerated wound closure, re-epithelialization and the suppression of Smad proteins (p-Smad2/3, Smad3). The data revealed that the suppression of Smad3, which was induced by bio-active ceramic resources powder particles affected re-epithelialization and cutaneous wound closure. At the end of this paper, we concluded that bio-active ceramic resources affect cutaneous wound healing by accelerating the re-epithelialization of keratinocytes and that is mediated by the suppression of related protein, Smad3.     

Inhibiting the inhibitor: the role of RNF12 in TGF-β superfamily signaling

In this issue of Molecular Cell, Zhang et al. (2012) identify the E3 ubiquitin ligase RNF12 as a new component of the TGF-β superfamily signaling pathways, which functions by targeting the negative regulator Smad7 for proteasomal degradation and thus potentiates pathway activity.     

Evolution of the TGF-β signaling pathway and its potential role in the ctenophore, Mnemiopsis leidyi

The TGF-β signaling pathway is a metazoan-specific intercellular signaling pathway known to be important in many developmental and cellular processes in a wide variety of animals. We investigated the complexity and possible functions of this pathway in a member of one of the earliest branching metazoan phyla, the ctenophore Mnemiopsis leidyi. A search of the recently sequenced Mnemiopsis genome revealed an inventory of genes encoding ligands and the rest of the components of the TGF-β superfamily signaling pathway. The Mnemiopsis genome contains nine TGF-β ligands, two TGF-β-like family members, two BMP-like family members, and five gene products that were unable to be classified with certainty. We also identified four TGF-β receptors: three Type I and a single Type II receptor. There are five genes encoding Smad proteins (Smad2, Smad4, Smad6, and two Smad1s). While we have identified many of the other components of this pathway, including Tolloid, SMURF, and Nomo, notably absent are SARA and all of the known antagonists belonging to the Chordin, Follistatin, Noggin, and CAN families. This pathway likely evolved early in metazoan evolution as nearly all components of this pathway have yet to be identified in any non-metazoan. The complement of TGF-β signaling pathway components of ctenophores is more similar to that of the sponge, Amphimedon, than to cnidarians, Trichoplax, or bilaterians. The mRNA expression patterns of key genes revealed by in situ hybridization suggests that TGF-β signaling is not involved in ctenophore early axis specification. Four ligands are expressed during gastrulation in ectodermal micromeres along all three body axes, suggesting a role in transducing earlier maternal signals. Later expression patterns and experiments with the TGF-β inhibitor SB432542 suggest roles in pharyngeal morphogenesis and comb row organization.     

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.     

Pathogenic role of TGF-β in the progression of podocyte diseases

In patients with progressive podocyte diseases, such as focal segmental glomerulosclerosis and membranous nephropathy, there is enhanced expression of transforming growth factor (TGF-β) in podocytes. Biomechanical strain in these diseases may cause overexpression of TGF-β and angiotensin II (Ang II) by podocytes. Oxidative stress induced by Ang II may activate the latent TGF-β. Increased TGF-β activity by podocytes may induce not only the thickening of the glomerular basement membrane (GBM), but also podocyte apoptosis and/or detachment from the GBM, initiating the development of glomerulosclerosis. Furthermore, mesangial matrix expansion frequently occurs in podocyte diseases in association with the development of glomerulosclerosis. This review examines open questions on the pathogenic role of TGF-β that links podocyte injury to GBM thickening, podocyte loss, mesangial matrix expansion and glomerulosclerosis in podocyte diseases. It also describes paracrine regulatory mechanisms of podocyte TGF-β on mesangial cells leading to increased matrix synthesis.     

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.     

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.     

Negative regulation of TGF-β signaling in development

The TGF-β superfamily members have important roles in controlling patterning and tissue formation in both invertebrates and vertebrates. Two types of signal transducers, receptors and Smads, mediate the signaling to regulate expression of their target genes. Despite of the relatively simple signal transduction pathway, many modulators have been found to contribute to a tight regulation of this pathway in a variety of mechanisms. This article reviews the negative regulation of TGF-β signaling with focus on its roles in vertebrate development.     

TGF-β1 signaling plays a dominant role in the crosstalk between TGF-β1 and the aryl hydrocarbon receptor ligand in prostate epithelial cells

Crosstalk between the aryl hydrocarbon receptor (AhR) and transforming growth factor-β1 (TGF-β1) signaling has been observed in various experimental models. However, both molecular mechanism underlying this crosstalk and tissue-specific context of this interaction are still only partially understood. In a model of human non-tumorigenic prostate epithelial cells BPH-1, derived from the benign prostatic hyperplasia, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) persistently activates the AhR signaling pathway and induces expression of xenobiotic metabolizing enzymes, such as CYP1A1 or CYP1B1. Here we demonstrate that TGF-β1 suppresses the AhR-mediated gene expression through multiple mechanisms, involving inhibition of AhR expression and down-regulation of nuclear AhR, via a SMAD4-dependent pathway. In contrast, TCDD-induced AhR signaling does not affect either TGF-β1-regulated gene expression or epithelial-to-mesenchymal transition. These observations suggest that, in the context of prostate epithelium, TGF-β1 signaling plays a dominant role in the crosstalk with AhR signaling pathway. Given the importance of TGF-β1 signaling in regulation of prostate epithelial tissue homeostasis, as well as the recently revealed role of AhR in prostate development and tumorigenesis, the above findings contribute to our understanding of the mechanisms underlying the crosstalk between the two signaling pathways in the prostate-specific context.     

The role of TGF-β in the pathogenesis of primary open-angle glaucoma

Transforming growth factor-β2 (TGF-β2) is found in increasing amounts in aqueous humor and reactive optic nerve astrocytes of patients with primary open-angle glaucoma (POAG), a major cause of blindness worldwide. The available data strongly indicate that TGF-β2 is a key player contributing to the structural changes in the extracellular matrix (ECM) of the trabecular meshwork and optic nerve head as characteristically seen in POAG. The changes involve an induction in the expression of various ECM molecules and are remarkably similar in trabecular meshwork cells and optic nerve head astrocytes. The ECM changes in the trabecular meshwork most probably play a role in the increase of aqueous humor outflow resistance causing higher intraocular pressure (IOP). In the optic nerve head, TGF-β2-induced changes might contribute to deformation of the optic nerve axons causing impairment of axonal transport and neurotrophic supply and leading to their continuous degeneration. The increase in IOP further adds mechanical stress and strain to optic nerve axons and accelerates degenerative changes. In addition, high IOP might induce the expression of activated TGF-β1 in trabecular meshwork cells and optic nerve head astrocytes; this again might significantly lead to the progress of axonal degeneration. The action of TGF-β2 in POAG is largely mediated through the connective tissue growth factor, whereas the activities of TGF-β1 and -β2 are modulated by the blocking effects of bone morphogenetic protein-4 (BMP-4) and BMP-7, by gremlin that inhibits BMP signaling and by several species of microRNAs.