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.     

Transforming growth factor β induces expression of connective tissue growth factor in hepatic progenitor cells through Smad independent signaling

Hepatic progenitor cells (HPCs) are activated in the chronic liver injury and are found to participate in the progression of liver fibrosis, while the precise role of HPCs in liver fibrosis remains largely elusive. In this study, by immunostaining of human liver sections, we confirmed that HPCs were activated in the cirrhotic liver and secreted transforming growth factor β (TGF-β) and connective tissue growth factor (CTGF), both of which were important inducers of liver fibrosis. Besides, we used HPC cell lines LE/6 and WB-F344 as in vitro models and found that TGF-β induced secretion of CTGF in HPCs. Moreover, TGF-β signaling was intracrine activated and contributed to autonomous secretion of CTGF in HPCs. Furthermore, we found that TGF-β induced expression of CTGF was not mediated by TGF-β activated Smad signaling but mediated by TGF-β activated Erk, JNK and p38 MAPK signaling. Taken together, our results provide evidence for the role of HPCs in liver fibrosis and suggest that the production of CTGF by TGF-β activated MAPK signaling in HPCs may be a therapeutic target of liver fibrosis.     

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.     

Integrin-mediated regulation of TGFβ in fibrosis

Fibrosis is a major cause of morbidity and mortality worldwide. Currently, therapeutic options for tissue fibrosis are severely limited, and organ transplantation is the only effective treatment for end-stage fibrotic disease. However, demand for donor organs greatly outstrips supply, and so effective anti-fibrotic treatments are urgently required. In recent years, the integrin family of cell adhesion receptors has gained prominence as key regulators of chronic inflammation and fibrosis. Fibrosis models in multiple organs have demonstrated that integrins have profound effects on the fibrotic process. There is now abundant in vivo data demonstrating critical regulatory roles for integrins expressed on different cell types during tissue fibrogenesis. In this review, we will examine the ways in which integrins regulate these processes and discuss how the manipulation of integrins using function blocking antibodies and small molecule inhibitors may have clinical utility in the treatment of patients with a broad range of fibrotic diseases. This article is part of a Special Issue entitled: Fibrosis: Translation of basic research to human disease.     

The role of transforming growth factor (TGF)-β in modulating the immune response and fibrogenesis in the gut

Transforming growth factor (TGF)-β, a pleiotropic cytokine released by both immune and non-immune cells in the gut, exerts an important tolerogenic action by promoting regulatory T cell differentiation. TGF-β also enhances enterocyte migration and regulates extracellular matrix turnover, thereby playing a crucial role in tissue remodeling in the gut. In this review we describe the mechanisms by which abnormal TGF-β signaling impairs intestinal immune tolerance and tissue repair, thus predisposing to the onset of immune-mediated bowel disorders, such as inflammatory bowel disease and celiac disease. Additionally, we will discuss potential therapeutic strategies aiming at restoring physiologic TGF-β signaling in chronic intestinal diseases.     

Matrix-dependent perturbation of TGFβ signaling and disease

Transforming growth factor beta (TGFβ) is a multipotent cytokine that is sequestered in the extracellular matrix (ECM) through interactions with a number of ECM proteins. The ECM serves to concentrate latent TGFβ at sites of intended function, to influence the bioavailability and/or function of TGFβ activators, and perhaps to regulate the intrinsic performance of cell surface effectors of TGFβ signal propagation. The downstream consequences of TGFβ signaling cascades in turn provide feedback modulation of the ECM. This review covers recent examples of how genetic mutations in constituents of the ECM or TGFβ signaling cascade result in altered ECM homeostasis, cellular performance and ultimately disease, with an emphasis on emerging therapeutic strategies that seek to capitalize on this refined mechanistic understanding.     

Role of fibrillin-2 in the control of TGF-β activation in tumor angiogenesis and connective tissue disorders

Fibrillins constitute a family of large extracellular glycoproteins which multimerize to form microfibrils, an important structure in the extracellular matrix. It has long been assumed that fibrillin-2 was barely present during postnatal life, but it is now clear that fibrillin-2 molecules form the structural core of microfibrils, and are masked by an outer layer of fibrillin-1. Mutations in fibrillins give rise to heritable connective tissue disorders, including Marfan syndrome and congenital contractural arachnodactyly. Fibrillins also play an important role in matrix sequestering of members of the transforming growth factor-β family, and in context of Marfan syndrome excessive TGF-β activation has been observed. TGF-β activation is highly dependent on integrin binding, including integrin αvβ8 and αvβ6, which are upregulated upon TGF-β exposure. TGF-β is also involved in tumor progression, metastasis, epithelial-to-mesenchymal transition and tumor angiogenesis. In several highly vascularized types of cancer such as hepatocellular carcinoma, a positive correlation was found between increased TGF-β plasma concentrations and tumor vascularity. Interestingly, fibrillin-1 has a higher affinity to TGF-β and, therefore, has a higher capacity to sequester TGF-β compared to fibrillin-2. The previously reported downregulation of fibrillin-1 in tumor endothelium affects the fibrillin-1/fibrillin-2 ratio in the microfibrils, exposing the normally hidden fibrillin-2. We postulate that fibrillin-2 exposure in the tumor endothelium directly stimulates tumor angiogenesis by influencing TGF-β sequestering by microfibrils, leading to a locally higher active TGF-β concentration in the tumor microenvironment. From a therapeutic perspective, fibrillin-2 might serve as a potential target for future anti-cancer therapies.     

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.     

IL-13 induces connective tissue growth factor in rat hepatic stellate cells via TGF-β-independent Smad signaling

Connective tissue growth factor (CTGF) plays a central role in stimulating extracellular matrix deposition in the liver, and hence is considered a critical mediator of TGF-β-dependent fibrogenesis. Hepatic stellate cells (HSCs) are known as the major source of CTGF in damaged liver. However, previous studies revealed that IL-13, rather than TGF-β, represents the predominant inducer of CTGF expression in HSCs. We now dissected IL-13 downstream signaling that modulates CTGF expression in HSCs. IL-13 induces a time- and dosage-dependent increase of CTGF in a TGF-β-independent manner. This process requires participation of different Smad proteins and their upstream receptor kinases (activin receptor-like kinases). Smad1 and Smad2 were identified as the key mediators of IL-13-dependent CTGF expression. Furthermore, IL-13 induces Stat6 phosphorylation in HSCs, but Stat6 was not involved in CTGF induction. Instead, the Erk1/2-MAPK pathway was found to be responsible for IL-13-induced early Smad phosphorylation and CTGF synthesis. We demonstrate that IL-13 induces CTGF expression in HSCs by activating TGF-β-independent activin receptor-like kinase/Smad signaling via the Erk-MAPK pathway rather than via its canonical JAK/Stat6 pathway. These results provide an improved new insight into the molecular mechanisms of profibrotic IL-13 activities in the liver.     

Oral glucosamine increases expression of transforming growth factor β1 (TGFβ1) and connective tissue growth factor (CTGF) mRNA in rat cartilage and kidney: implications for human efficacy and toxicity

Glucosamine is used for alleviating pain in osteoarthritis. Clinical trials have reported that glucosamine has equivocal efficacy. Glucosamine is also used in cell cultures to stimulate hexosamine flux and protein O-glycosylation, but at many-fold greater concentrations than those in human plasma following oral dosing. Lean Zucker rats were dosed orally for 6 weeks with glucosamine hydrochloride at doses (0–600 mg/kg/day) that produced peak serum concentrations of <1–35 μM, spanning the human exposure range. Relative expression of both TGFβ1 and CTGF mRNA were significantly increased up to 2.3-fold in liver, kidney and articular cartilage when evaluated 4 h after final dose. Apparent threshold serum glucosamine (C_max) concentration required to increase TGFβ1 expression in cartilage was 10–20 μM. These increases were associated with significant increases in UDP-N-acetylglucosamine concentrations suggesting increased hexosamine flux. Both TGFβ1 and CTGF are mediators of chondrocyte proliferation and cartilage repair. Study demonstrates that oral glucosamine doses that produce clinically relevant serum glucosamine concentrations can induce tissue TGFβ1 and CTGF expression in vivo and provides a mechanistic rationale for reported beneficial effects of glucosamine therapy. Induction of renal TGFβ1 and CTGF mRNA suggests that potential sclerotic side-effects may occur following consumption of potent glucosamine preparations.     

TGFβ superfamily signaling in the neural crest lineage

The neural crest cell (NCC) lineage is often referred to as the fourth germ layer in embryos, as its wide range of migration and early colonization of multiple tissues and organ systems throughout the developing body is astounding. Many human birth defects are thought to have their origins within the NCC lineage. Exciting recent conditional mouse targeting and transgenic combinatorial suppression approaches have revealed that the Tgf-b superfamily is a key signaling pathway within the cardiac and cranial NCC subpopulations. Given the complexity of Tgf-b superfamily signaling and that multiple ligand and receptor combinations have already been shown to be expressed within the NCC subpopulations, and the difficulty in transgenically targeting entire signaling cascades, we review several up-to-date transgenic approaches that are revealing unexpected consequences.     

TGFβ superfamily signaling in the neural crest lineage

The neural crest cell (NCC) lineage is often referred to as the fourth germ layer in embryos, as its wide range of migration and early colonization of multiple tissues and organ systems throughout the developing body is astounding. Many human birth defects are thought to have their origins within the NCC lineage. Exciting recent conditional mouse targeting and transgenic combinatorial suppression approaches have revealed that the TGFβ superfamily is a key signaling pathway within the cardiac and cranial NCC subpopulations. Given the complexity of TGFβ superfamily signaling and that multiple ligand and receptor combinations have already been shown to be expressed within the NCC subpopulations, and the difficulty in transgenically targeting entire signaling cascades, we review several up-to-date transgenic approaches that are revealing unexpected consequences.Key words: TGFβ, neural crest, heart, cranial crest, mouse transgenics     

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.     

What's the place of immunotherapy in malignant mesothelioma treatments?

Malignant pleural mesothelioma (MPM) is a rare malignancy of the pleura with a very poor prognosis. Treatments evaluated for malignant mesothelioma, including chemotherapy, radiotherapy and surgery are of limited efficacy. However, the fact that the tumors of some patients with MPM regress spontaneously or respond to immunotherapy suggests that the immune system may respond to MPM under some circumstances. In this respect, animal studies have demonstrated immunoreactivity of MPM to different immunotherapies. In the case of MPM, several clinical studies have demonstrated a correlation between the presence of a lymphocyte infiltrate and a better prognosis and humoral response directed against specific antigens related to tumor. Thus, MPM immunotherapy is undoubtedly a highly promising but also very challenging approach to the treatment of this disease that has slipped through the defense lines of the immune system. This article reviews past and recent developments of the clinical strategies that concern immunotherapy of mesothelioma.