Multifaceted transforming growth factor-beta (TGFβ) signalling in glioblastoma

Glioblastoma (GBM) is an aggressive and devastating primary brain cancer which responds very poorly to treatment. The average survival time of patients is only 14–15 months from diagnosis so there is a clear and unmet need for the development of novel targeted therapies to improve patient outcomes. The multifunctional cytokine TGFβ plays fundamental roles in development, adult tissue homeostasis, tissue wound repair and immune responses. Dysfunction of TGFβ signalling has been implicated in both the development and progression of many tumour types including GBM, thereby potentially providing an actionable target for its treatment. This review will examine TGFβ signalling mechanisms and their role in the development and progression of GBM. The targeting of TGFβ signalling using a variety of approaches including the TGFβ binding protein Decorin will be highlighted as attractive therapeutic strategies.     

Proteomic identification of ADAM12 as a regulator for TGF-β1-induced differentiation of human mesenchymal stem cells to smooth muscle cells

Background

Transforming growth factor-β1 (TGF-β1) induces the differentiation of human adipose tissue-derived mesenchymal stem cells (hASCs) into smooth muscle cells. Lipid rafts are cholesterol-rich microdomains in cell membranes that reportedly play a key role in receptor-mediated signal transduction and cellular responses. In order to clarify whether lipid rafts are involved in TGF-β1-induced differentiation of hASCs into smooth muscle cells, we analyzed the lipid raft proteome of hASCs.

Methods and Results

Pretreatment of hASCs with the lipid raft disruptor methyl-β-cyclodextrin abrogated TGF-β1-induced expression of α-smooth muscle actin, a smooth muscle cell marker, suggesting a pivotal role of lipid rafts in TGF-β1-induced differentiation of hASCs to smooth muscle cells. Sucrose density gradient centrifugation along with a shotgun proteomic strategy using liquid chromatography-tandem mass spectrometry identified 1002 individual proteins as the lipid raft proteome, and 242 of these were induced by TGF-β1 treatment. ADAM12, a disintegrin and metalloproteases family member, was identified as the most highly up-regulated protein in response to TGF-β1 treatment. TGF-β1 treatment of hASCs stimulated the production of both ADAM12 protein and mRNA. Silencing of endogenous ADAM12 expression using lentiviral small hairpin RNA or small interfering RNA abrogated the TGF-β1-induced differentiation of hASCs into smooth muscle cells.

Conclusions

These results suggest a pivotal role for lipid raft-associated ADAM12 in the TGF-β1-induced differentiation of hASCs into smooth muscle cells.     

Color-coded intravital imaging demonstrates a transforming growth factor-β (TGF-β) antagonist selectively targets stromal cells in a human pancreatic-cancer orthotopic mouse model

Pancreatic cancer is a recalcitrant malignancy, partly due to desmoplastic stroma which stimulates tumor growth, invasion, and metastasis, and inhibits chemotherapeutic drug delivery. Transforming growth factor-β (TGF-β) has an important role in the formation of stromal desmoplasia. The present study describes the ability of color-coded intravital imaging to demonstrate the efficacy of a TGF-β inhibitor to target stroma in an orthotopic mouse model of pancreatic cancer. The BxPC-3 human pancreatic adenocarcinoma cell line expressing green fluorescent protein (GFP), which also has a high TGF-β expression level, was used in an orthotopic model in transgenic nude mice ubiquitously expressing red fluorescent protein (RFP). Fourteen mice were randomized into a control group (n = 7, vehicle, i.p., weekly, for 3 weeks) and a treated group (n = 7, SB431542 [TGF-β receptor type I inhibitor] 0.3 mg, i.p., weekly, for 3 weeks). Stromal cells expressing RFP and cancer cells expressing GFP were observed weekly for 3 weeks by real-time color-coded intravital imaging. The RFP fluorescence area from the stromal cells, relative to the GFP fluorescence area of the cancer cells, was significantly decreased in the TGF-β-inhibitor-treatment group compared to the control group. The present study demonstrated color-coded imaging in an orthotopic pancreatic-cancer cell-line mouse model can readily detect the selective anti-stromal-cell targeting of a TGF-β inhibitor.     

Induction of apoptosis in bacillus Calmette-Guérin-activated T cells by transforming growth factor-beta

In view of the critical role played by bacillus Calmette-Guérin (BCG) in the development and functional activation of protective T cells against tuberculosis, it has become important to understand the mechanisms by which cytokines regulate BCG-mediated immune responses. There is evidence that cytokine-mediated suppression of T cell function by mechanisms, including apoptosis, may reduce host resistance in tuberculosis. However, it is unclear whether cytokine-mediated suppression of antigen-responsive T cells through apoptotic mechanisms may be operating during human cellular activation induced by BCG. Here we present evidence, for the first time, that treatment of BCG-activated T cells with transforming growth factor-beta (TGF-beta) induces cellular apoptosis. These results were further supported by the fact that treatment of cells with a blocking mAb directed to TGF-beta significantly inhibited the percentage of apoptosis induced by TGF-beta. Interestingly, TGF-beta-mediated death of BCG-activated T cells in cultures containing interleukin (IL)-12 was observed. Moreover, our results demonstrated the induction of apoptosis by TGF-beta in BCG-activated T cells cultured in the presence of exogenous IL-12. In addition, our data indicated that TGF-beta significantly inhibited both BCG-induced cell growth determined by thymidine uptake and BCG-induced IFN-gamma secretion. Finally, TGF-beta-induced apoptosis in BCG-activated T cells correlated inversely with BCG-induced IFN-gamma secretion. Taken together, these findings indicate that TGF-beta induces apoptosis in human T cells activated with BCG and at the same time suggest that loss of BCG-reactive T cells through apoptotic mechanisms could contribute to an increased susceptibility to Mycobacterium tuberculosis infection.     

TGF-β regulates β-catenin signaling and osteoblast differentiation in human mesenchymal stem cells

Human adult bone marrow-derived skeletal stem cells a.k.a mesenchymal stem cells (hMSCs) have been shown to be precursors of several different cellular lineages, including osteoblast, chondrocyte, myoblast, adipocyte, and fibroblast. Several studies have shown that cooperation between transforming growth factor β (TGF-β) and Wnt/β-catenin signaling pathways plays a role in controlling certain developmental events and diseases. Our previous data showed that agents like TGF-β, cooperation with Wnt signaling, promote chondrocyte differentiation at the expense of adipocyte differentiation in hMSCs. In this study, we tested mechanisms by which TGF-β activation of β-catenin signaling pathway and whether these pathways interact during osteoblast differentiation of hMSCs. With selective small chemical kinase inhibitors, we demonstrated that TGF-β1 requires TGF-β type I receptor ALK-5, Smad3, phosphoinositide 3-kinases (PI3K), and protein kinase A (PKA) to stabilize β-catenin, and needs ALK-5, PKA, and JNK to inhibit osteoblastogenesis in hMSCs. Knockdown of β-catenin with siRNA stimulated alkaline phosphatase activity and antagonized the inhibitory effects of TGF-β1 on bone sialoprotein (BSP) expression, suggested that TGF-β1 cooperated with β-catenin signaling in inhibitory of osteoblastogenesis in hMSCs. In summary, TGF-β1 activates β-catenin signaling pathway via ALK-5, Smad3, PKA, and PI3K pathways, and modulates osteoblastogenesis via ALK5, PKA, and JNK pathways in hMSCs; the interaction between TGF-β and β-catenin signaling supports the view that β-catenin signaling is a mediator of TGF-β's effects on osteoblast differentiation of hMSCs.     

Synthesis of embryonic tendon-like tissue by human marrow stromal/mesenchymal stem cells requires a three-dimensional environment and transforming growth factor β3

Tendon-like tissue generated from stem cells in vitro has the potential to replace tendons and ligaments lost through injury and disease. However, thus far, no information has been available on the mechanism of tendon formation in vitro and how to accelerate the process. We show here that human mesenchymal stem cells (MSCs) and bone marrow-derived mononuclear cells (BM-MNCs) can generate tendon-like tissue in 7 days mediated by transforming growth factor (TGF) β3. MSCs cultured in fixed-length fibrin gels spontaneously synthesized narrow-diameter collagen fibrils and exhibited fibripositors (actin-rich, collagen fibril-containing plasma membrane protrusions) identical to those that occur in embryonic tendon. In contrast, BM-MNCs did not synthesize tendon-like tissue under these conditions. We performed real-time PCR analysis of MSCs and BM-MNCs. MSCs upregulated genes encoding type I collagen, TGFβ3, and Smad2 at the time of maximum contraction of the tendon-like tissue (7 days). Western blot analysis showed phosphorylation of Smad2 at maximum contraction. The TGFβ inhibitor SB-431542, blocked the phosphorylation of Smad2 and stopped the formation of tendon-like tissue. Quantitative PCR showed that BM-MNCs expressed very low levels of TGFβ3 compared to MSCs. Therefore we added exogenous TGFβ3 protein to BM-MNCs in fibrin gels, which resulted in phosphorylation of Smad2, synthesis of collagen fibrils, the appearance of fibripositors at the plasma membrane, and the formation of tendon-like tissue. In conclusion, MSCs that self-generate TGFβ signaling or the addition of TGFβ3 protein to BM-MNCs in fixed-length fibrin gels spontaneously make embryonic tendon-like tissue in vitro within 7 days.     

Culture of human mesenchymal stem cells on microcarriers in a 5 l stirred-tank bioreactor

For the first time, fully functional human mesenchymal stem cells (hMSCs) have been cultured at the litre-scale on microcarriers in a stirred-tank 5 l bioreactor, (2.5 l working volume) and were harvested via a potentially scalable detachment protocol that allowed for the successful detachment of hMSCs from the cell-microcarrier suspension. Over 12 days, the dissolved O₂ concentration was >45 % of saturation and the pH between 7.2 and 6.7 giving a maximum cell density in the 5 l bioreactor of 1.7 × 10⁵ cells/ml; this represents >sixfold expansion of the hMSCs, equivalent to that achievable from 65 fully-confluent T-175 flasks. During this time, the average specific O₂ uptake of the cells in the 5 l bioreactor was 8.1 fmol/cell h and, in all cases, the 5 l bioreactors outperformed the equivalent 100 ml spinner-flasks run in parallel with respect to cell yields and growth rates. In addition, yield coefficients, specific growth rates and doubling times were calculated for all systems. Neither the upstream nor downstream bioprocessing unit operations had a discernible effect on cell quality with the harvested cells retaining their immunophenotypic markers, key morphological features and differentiation capacity.     

Effects of transforming growth factor β on growth of human mammary epithelial cells in culture

Summary Normal human mammary epithelial cells (HMEC) from different individual reduction mammoplasty specimens were all growth inhibited, and showed a flattened, elongated morphology in response to human recombinant transforming growth factor β1 (TGFβ). The degree of growth inhibition varied among specimens, but none of the normal HMEC maintained growth in the continued presence of TGFβ. The degree of growth inhibition also varied with cell age in vitro, cells closer to senescence being more sensitive. TGFβ sensitivity was additionally assayed in two established cell lines derived from one of the reduction mammoplasty specimens after exposure to benzo(a)pyrene. Although varying degrees of growth inhibition and morphologic changes were observed in the cell lines, both lines contained populations that maintained active growth in the presence of TGFβ. Subclones of these lines demonstrated a great plasticity in their growth response to TGFβ, with individual clones ranging from strongly growth inhibited to nearly unaffected. These results suggest that multiple factors influence the extent of TGFβ-induced growth effects on both normal and transformed mammary epithelial cells, and that some of these factors may act through epigenetic mechanisms. This work was supported by CA24844 from the National Institutes of Health, Bethesda, MD, and the Office of Energy Research, Office of Health and Environmental Research of the U.S. Department of Energy under contract DE-AC03-76SF00098.     

Does transforming growth factor-beta (TGF-beta) act as a neuroprotective agent in cerebral ischemia?

Necrosis and apoptosis are the two fundamental hallmarks of neuronal death in stroke. Nevertheless, thrombolysis, by means of the recombinant serine protease t-PA, remains until now the only approved treatment of stroke in man. Over the last years, the cytokine termed Transforming Growth Factor-beta 1 (TGF-beta 1) has been found to be strongly up regulated in the central nervous system following ischemia-induced brain damage. Recent studies have shown a neuroprotective activity of TGF-beta 1 against ischemia-induced neuronal death. In vitro, TGF-beta 1 protects neurons against excitotoxicity by inhibiting the t-PA-potentiated NMDA-induced neuronal death through a mechanism involving the up-regulation of the type-1 plasminogen activator inhibitor (PAI-1) in astrocytes. Altogether, these observations suggest that either TGF-beta signaling or TGF-beta 1-modulated genes could be good targets for the development of new therapeutic strategies for stroke in man.     

Relative mRNA expression and immunolocalization for transforming growth factor-beta (TGF-β) and their effect on in vitro development of caprine preantral follicles

This study aimed to evaluate the immunolocalization and messenger RNA (mRNA) expression for transforming growth factor-beta (TGF-β) and its receptors (TGF-βRI and RII), as well as mRNA expression for P450 aromatase and FSH receptor in caprine preantral follicles. The effects of TGF-β, FSH alone, or in association on the in vitro follicular development were also assessed. Immunohistochemical analyses showed the expression of TGF-β and its receptors in oocytes of all follicle stages and granulosa cells of primary and secondary follicles. mRNA for TGF-β receptors and for FSH receptor (FSHR) was present in preantral follicles as well as in oocytes and granulosa cells of antral follicles. Isolated secondary follicles were cultured in α-minimum essential medium (MEM) alone or supplemented with either FSH (100 ng/ml), TGF-β (10 ng/ml), or TGF-β + FSH for 18 d. TGF-β increased significantly oocyte diameter when compared to FSH alone and control. After 18 d of culture, all groups showed a significant reduction in P450 aromatase and FSHR mRNA levels in comparison to fresh control. In contrast, treatment with FSH significantly increased the mRNA expression for TGF-β in comparison to fresh control and other treatments. In conclusion, the findings showed that TGF-β and its receptors are present in caprine ovarian follicles. Furthermore, they showed a positive effect on oocyte growth in vitro.     

Regulation of autophagy by transforming growth factor-β (TGF-β) signaling

Transforming growth factor-β (TGF-β) has broad impacts on an array of diverse cellular functions including cell growth, differentiation, adhesion, migration, and apoptosis. Perturbations of the TGF-β signaling pathways are involved in progression of various tumors. Autophagy is a pivotal response of normal and cancer cells to environmental stresses and is induced by various stimuli. Otherwise, autophagy has an intrinsic function in tumor suppression. Recently, we demonstrated that TGF-β induces autophagy in hepatocellular carcinoma cells and mammary carcinoma cells. Autophagy activation by TGF-β is mediated through the Smad and JNK pathways. We show that siRNA-mediated knockdown of autophagy genes suppresses the growth inhibitory function of TGF-β and that autophagy activation potentiates TGF-β-mediated induction of proapoptotic genes, Bim and Bmf, in hepatoma cells. In this context, the autophagy pathway might contribute to the growth inhibitory effect of TGF-β, in conjunction with other anti-proliferative pathways downstream of TGF-β signaling. The context and manner by which the TGF-β signaling pathway regulates autophagy have implications for a better understanding of pathological and bidirectional roles of TGF-β signaling pathways in tumorigenesis.     

Thrombospondin-1 inhibits osteogenic differentiation of human mesenchymal stem cells through latent TGF-β activation

Transforming growth factor-β (TGF-β) is a critical regulator of bone development and remodeling. TGF-β must be activated from its latent form in order to signal. Thrombospondin-1 (TSP1) is a major regulator of latent TGF-β activation and TSP1 control of TGF-β activation is critical for regulation of TGF-β activity in multiple diseases. Bone marrow-derived mesenchymal stem cells (MSCs) have osteogenic potential and they participate in bone remodeling in injury and in response to tumor metastasis. Since both TSP1 and TGF-β inhibit osteoblast differentiation, we asked whether TSP1 blocks osteoblast differentiation of MSCs through its ability to stimulate TGF-β activation. TSP1 added to human bone marrow-derived MSCs under growth conditions increases active TGF-β. Cultured MSCs express TSP1 and both TSP1 expression and TGF-β activity decrease during osteoblast differentiation. TSP1 and active TGF-β block osteoblast differentiation of MSCs grown in osteogenic media as measured by decreased Runx2 and alkaline phosphatase expression. The inhibitory effect of TSP1 on osteoblast differentiation is due to its ability to activate latent TGF-β, since a peptide which blocks TSP1 TGF-β activation reduced TGF-β activity and restored osteoblast differentiation as measured by increased Runx2 and alkaline phosphatase expression. Anti-TGF-β neutralizing antibody also increased alkaline phosphatase expression in the presence of TSP1. These studies show that TSP1 regulated TGF-β activity is a critical determinant of osteoblast differentiation.     

Characterization of a human hepatoma cell line with acquired resistance to growth inhibition by transforming growth factor beta 1 (TGF-β1)

Summary A new cell line (Hep 3B-TR), which is resistant to growth-inhibition by transforming growth factor beta 1 (TGF-β1) up to 10 ng/ml (400 pM), was isolated from parental Hep 3B human hepatoma cells, which are sensitive to growth-inhibition by TGF-β1. In the presence of TGF-β1 (1 to 10 ng/ml), the growth of the parental cell line (Hep 3B-TS) was inhibited by more than 95%. Under the same conditions, the growth rate of the resistant clone (Hep 3B-TR) however, was identical in the presence or absence of TGF-β1 and was almost the same as that of the Hep 3B-TS cells in the absence of TGF-β1. Affinity crosslinking with 5 pM ¹²⁵I-labeled TGF-β1 showed that the TGF-β1 receptors type I (TGF-βRI) and type II (TGF-βRII) were not present on the cell surface of the Hep 3B-TR cells, whereas they were present on the sensitive HEP 3B-TS cells. Hep 3B-TS cells had detectable TGF-βRII mRNA, which was not found in Hep 3B-TR cells. RNA analysis showed different effects on the expression of TGF-β1, c-fos, c-myc, and protein disulfide isomerase (PDI) genes in the two cell lines in response to TGF-β1 protein. Addition of TGF-β1 (1 ng/ml) strongly increased the expression of TGF-β1 mRNA in Hep 3B-TS cells, but not in Hep 3B-TR cells. In Hep 3B-TS cells, c-fos mRNA was not detected either in the presence or absence of TGF-β1 protein. However, abundant c-fos mRNA was detected in Hep 3B-TR cells, which was not altered by TGF-β1 protein. TGF-β1 protein inhibited the expression of c-myc and PDI mRNAs in Hep 3B-TS cells, whereas although the c-myc and PDI mRNAs were much more abundant in Hep 3B-TR cells, their expression was not affected by TGF-β1 protein. These results suggest that the mechanisms of escape from growth-inhibition by TGF-β1 in Hep 3B-TR hepatoma cells probably involve loss of binding by TGF-β1 to its cell surface receptors.     

Alteration of protein glycosylation in human hepatic stellate cells activated with transforming growth factor-β1

Although aberrant glycosylation of human glycoproteins is related to liver fibrosis that results from chronic damage to the liver in conjunction with the activation of hepatic stellate cells (HSCs), little is known about the precision alteration of protein glycosylation referred to the activation of HSCs by transforming growth factor-β1 (TGF-β1). The human HSCs, LX-2 were activated by TGF-β1. The lectin microarrays were used to probe the alteration of protein glycosylation in the activated HSCs compared with the quiescent HSCs. Lectin histochemistry was used to further validate the lectin binding profiles and assess the distribution of glycosidic residues in cells. As a result, 14 lectins (e. g. AAL, PHA-E, ECA and ConA) showed increased signal while 7 lectins (e. g. UEA-I and GNA) showed decreased signal in the activated LX-2 compared with the quiescent LX-2. Meanwhile, AAL, PHA-E and ECA staining showed moderate binding to the cytoplasma membrane in the quiescent LX-2, and the binding intensified in the same regions of the activated LX-2. In conclusion, the precision alteration of protein glycosylation related to the activation of the HSCs may provide useful information to find new molecular mechanism of HSC activation and antifibrotic therapeutic strategies.