Differential in vitro phenotype pattern, transforming growth factor-β1 activity and mRNA expression of transforming growth factor-β1 in Apert osteoblasts

The phenotype of Apert osteoblasts differs from that of normal osteoblasts in the accumulation of macromolecules in the extracellular matrix. Apert osteoblasts increase type I collagen, fibronectin and glycosaminoglycans secretion compared with normal osteoblasts. Because the extracellular matrix macromolecule accumulation is greatly modulated by transforming growth factor-beta(1), we examined the ability of normal and Apert osteoblasts to secrete transforming growth factor-beta(1) by CCL-64 assay and to produce transforming growth factor-beta(1 )by analysis of the mRNA expression of transforming growth factor-beta(1). Northern blot analysis revealed an increased amount of transforming growth factor-beta(1) mRNA expression in Apert osteoblasts compared with normal ones. Moreover, the level of the active transforming growth factor-beta(1) isoform was higher in Apert than in normal media. In pathologic cells, the increase in transforming growth factor-beta(1) gene expression was associated with a parallel increase in the factor secreted into the medium. The level of transforming growth factor-beta(1) was decreased by the addition of basic fibroblast growth factor. Transforming growth factor-beta(1) is controlled temporally and spatially during skeletal tissue development and produces complex stimulatory and inhibitory changes in osteoblast functions. We hypothesise that in vitro differences between normal and Apert osteoblasts may be correlated to different transforming growth factor-beta(1) cascade patterns, probably due to an altered balance between transforming growth factor-beta(1) and basic fibroblast growth factor.     

Tumor necrosis factor-α and transforming growth factor-β1 facilitate differentiation and proliferation of tendon-derived stem cells in vitro

Objectives

To investigate the effects of tumor necrosis factor-α (TNF-α) and transforming growth factor-β1 (TGF-β1) on the proliferation and differentiation of tendon-derived stem cells (TDSC).

Results

TNF-α inhibits the proliferation and tenogenic/osteogenic differentiation of TDSC but, after simultaneous or sequential treatment with TGF-β1 and TNF-α, the expression of tenogenic/osteogenic-related marker and proliferation of TDSC was significantly increased. During these processes, Smad2/3 and Smad1/5/8 were highly phosphorylated, meaning that the TGF-β and BMP signaling pathways were highly activated. Further study revealed that the expression of Inhibitor-Smad appeared to be negatively correlated to the proliferation and differentiation of TDSC.

Conclusions

Combining the use of TNF-α and TGF-β1 could improve the proliferation and differentiation of TDSC in vitro, and the expression of I-Smad is negatively correlated with TDSC proliferation and differentiation.

     

Effect of transforming growth factor-β3 on mono and multilayer chondrocytes

Articular cartilage is an avascular and flexible connective tissue found in joints. It produces a cushioning effect at the joints and provides low friction to protect the ends of the bones from wear and tear/damage. It has poor repair capacity and any injury can result pain and loss of mobility. Transforming growth factor-beta (TGF-β), a cytokine superfamily, regulates cell function, including differentiation and proliferation. Although the function of the TGF-βs in various cell types has been investigated, their function in cartilage repair is as yet not fully understood. The effect of TGF-β3 in biological regulation of primary chondrocyte was investigated in this work. TGF-β3 provided fibroblastic morphology to chondrocytes and therefore overall reduction in cell proliferation was observed. The length of the cells supplemented with TGF-β3 were larger than the cells without TGF-β3 treatment. This was caused by the fibroblast like cells (dedifferentiated chondrocytes) which occupied larger areas compared to cells without TGF-β3 addition. The healing process of the model wound closure assay of chondrocyte multilayer was slowed down by TGF-β3, and this cytokine negatively affected the strength of chondrocyte adhesion to the cell culture surface.     

Modulation of YY1 and p53 expression by transforming growth factor-β3 in prostate cell lines

Transforming growth factor-β (TGF-β) is the prototype of a family of secreted polypeptide growth factors. These cytokines play very important roles during development, as well as in normal physiological and disease processes, by regulating a wide array of cellular processes, such as cell growth, differentiation, migration, apoptosis, and extracellular matrix production. TGF-β utilizes a multitude of intracellular signalling pathways in addition to Smads with actions that are dependent on circumstances, including dose, target cell type, and context. The aims of this research were (i) to verify the effects of dose-dependent TGF-β3 treatment on YY1 and p53 expression, in BPH-1 cell line, human benign prostate hyperplasia, and two prostate cancer cell lines, LNCaP, which is androgen-sensitive, and DU-145, which is androgen-non responsive, (ii) establish a correlation between p53 and YY1 and (iii) determine the expression of a number of important intracellular signalling pathways in TGF-β3-treated prostate cell lines. The expression of YY1, p53, PI3K, AKT, pAKT, PTEN, Bcl-2, Bax, and iNOS was evaluated through Western blot analysis on BPH-1, LNCaP, and DU-145 cultures treated with 10 and 50 ng/ml of TGF-β3 for 24 h. The production of nitric oxide (NO) was determined by Griess reagent and cell viability through MTT assay. The results of this research demonstrated profound differences in the responses of the BPH-1, LNCaP, and DU-145 cell lines to TGF-β3 stimulation. We believe that the findings could be important because of the clinical relevance that they may assume and the therapeutic implications for TGF-β treatment of prostate cancer.     

Clinical significance of serum transforming growth factor-β1 in lung cancer

Background: Transforming growth factor-β1 (TGF-β1) plays a critical role in human cancer development. Present study aimed to explore the clinical significance of serum TGF-β1 levels in patients with lung cancer and analyze the relationship between TGF-β1 and existing tumor markers for lung cancer. Methods: Serum was collected from 118 patients with lung cancer and 40 healthy volunteers. Serum TGF-β1 levels were measured by enzyme-linked immunosorbent assay (ELISA), and the association with various clinical characteristics was analyzed. The diagnostic value of TGF-β1 was assessed alone and in combination with existing tumor markers for lung cancer. Results: Serum TGF-β1 levels were significantly higher in patients with lung cancer compared to healthy volunteers [0.6 × 10⁵ (0.4 × 10⁵, 0.9 × 10⁵) pg/ml vs 0.5 × 10⁵ (0.3 × 10⁵, 0.7 × 10⁵) pg/ml, P = 0.040]. Although there was a positive correlation between serum TGF-β1 levels and advanced stages, the significant difference was not found between early stages and advanced stages (P = 0.116). The ability of serum TGF-β1 to discriminate lung cancer at a cutoff value of 79,168 pg/ml exhibited sensitivity of 30.6% and specificity of 97.5%. Serum TGF-β1 levels were correlated to cytokeratin fragment 21-1 (CYFRA21-1; R = 0.308, P = 0.020) and neuron-specific enolase (NSE; R = 0.558, P = 0.003). The diagnostic accuracy rates for the existing lung-tumor markers, as SCC, CYFRA21-1, and NSE, were increased from 20.0%, 34.6%, and 45.9% to 48.9%, 51.7%, and 54.5%, respectively by the inclusion of serum TGF-β1 levels. Conclusion: Quantification of serum TGF-β1 levels by ELISA may provide a novel complementary tool for the clinical diagnosis of lung cancer.     

Cellular origin and distribution of transforming growth factor-β1 in the normal rat myocardium

Summary Transforming growth factor- (TGF-) is a biologically active polypeptide present in normal tissues as well as transformed cells. Two structurally related forms of this peptide are TGF- ₁ and TGF- ₂. Using freshly isolated cardiomyocytes and non-myocyte heart cells, and a [³²P]-labelled cDNA probe to human TGF- ₁, we demonstrated that mRNA for TGF- ₁ could be detected only in the nonmyocyte fraction of heart cells. In the present study, the distribution of TGF- ₁ in the heart was determined by immunofluorescence staining by use of a polyclonal antibody to porcine TGF- ₁ in cryostat sections of rat heart. Immunofluorescence staining was intense around the blood vessels and radially diffuse in the surrounding myocardium.     

Growth inhibition induced by transforming growth factor-β1 in human oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is a world-wide health problem and its incidence accounts for 1.9–3.5% of all malignant tumors. Transforming growth factor beta/Smads (TGF-β/Smads) signaling pathway plays an important role in oncogenesis, but its function and molecular mechanisms in OSCC remain unclear. Expression of transforming growth factor-β receptor type II (TβRII) and Smad4 was studied by immunohistochemistry in 108 OSCC patients and 10 normal controls. Function and molecular mechanisms of TGF-β/Smads signaling pathway was then investigated in two human tongue squamous carcinoma cell lines with high and low metastasis (Tb and Tca8113) by RT-PCR, Western Blot, immunofluorescence, cell growth curve and flow cytometry (FCM), respectively. TβRII and Smad4 were significantly down-regulated in tumor tissues (with or without lymph node metastasis) compared to normal oral epithelium tissues (P < 0.05). TGF-β1 induced arrest of the cell cycle rather than cell death in Tca8113 and Tb cells, and this influence was mediated by the increasing the expression and changing the location of its downstream components of TGF-β1/Smads signaling pathway. TGF-β1 rapidly increased the expression of p15 and p21 in both Tca8113 and Tb cells. TGF-β1 did not increase p27 expression in Tca8113 cells, but p27 expression was increased in Tb cells. These indicated that TGF-β1 induced G₁ arrest of cell cycle through a different regulating pathway in Tb cells compared with Tca8113 cells. Thus, we conclude that TGF-β/Smads signaling pathway play a important role on cell growth and metastasis potential in OSCC. Xiumei Wang, Wenjing Sun, and Jing Bai contributed equally to this paper.     

Utility of plasma tumour necrosis factor-α and transforming growth factor-β1 as predictors of survival and treatment outcome in advanced non-small cell lung carcinoma

We hypothesized that plasma level of tumour necrosis factor (TNF)-α and transforming growth factor (TGF)-β1 may be a potential tool for diagnosis, prognosis and prediction of treatment outcome in non-small cell lung carcinoma (NSCLC). Plasma levels of TNF-α and TGF-β1 were quantified in 100 NSCLC patients and 100 controls. Association of TNF-α and TGF-β1 with response to therapy and survival was determined in 42 patients. An increased presence of TNF-α and TGF-β1 was observed in NSCLC compared with controls. TNF-α and TGF-β1 levels did not correlate with survival and response to chemotherapy. TNF-α and TGF-β1 do not appear to be reliable markers for predicting survival and response to therapy in advanced NSCLC.     

Effect of transforming growth factor-β on lipoprotein lipase in rat mesenchymal heart cell cultures

The effect of recombinant transforming growth factor-β2 (rTGF-β2) on lipoprotein lipase (LPL) synthesis was studied in mesenchymal rat heart cell cultures. Addition of rTGF-β2 to culture medium containing 20% serum resulted in a time-dependent decrease in LPL activity. With 10 ng/ml a 30% fall occurred after 12 h and only 20% of enzyme activity remained after 24 h with 5 or 10 ng/ml. The minimal effective dose of rTGF-β2 was 0.1 ng/ml and a 20% decrease occurred after exposure for 24 h. Antibodies specific to TGF-β2 blocked this effect. The decrease in enzymic activity was accompanied by a decrease in enzume mass and LPL mRNA. Addition of rTGF-β2 was effective only during the first week in culture, when enzyme activity was increasing but not after 12 days when the cultures were overconfluent, and the enzyme activity was high.     

Combined effects of interleukin-1α and transforming growth factor-β1 on modulation of human cardiac fibroblast function

During cardiac remodeling, cardiac fibroblasts (CF) are influenced by increased levels of interleukin-1α (IL-1α) and transforming growth factor-β1 (TGFβ1). The present study investigated the interaction between these two important cytokines on function of human CF and their differentiation to myofibroblasts (CMF). CF were isolated from human atrial appendage and exposed to IL-1α and/or TGFβ1 (both 0.1 ng/ml). mRNA expression levels of selected genes were determined after 6–24 h by real-time RT-PCR, while protein levels were analyzed at 24–48 h by ELISA or western blot. Activation of canonical signaling pathways (NFκB, Smad3, p38 MAPK) was determined by western blotting. Differentiation to CMF was examined by collagen gel contraction assays. Exposure of CF to IL-1α alone enhanced levels of IL-6, IL-8, matrix metalloproteinase-3 (MMP3) and collagen III (COL3A1), but reduced the CMF markers α-smooth muscle actin (αSMA) and connective tissue growth factor (CTGF/CCN2). By contrast, TGFβ1 alone had minor effects on IL-6, IL-8 and MMP3 levels, but significantly increased levels of the CMF markers αSMA, CTGF, COL1A1 and COL3A1. Co-stimulation with both IL-1α and TGFβ1 increased MMP3 expression synergistically. Furthermore, while TGFβ1 had no effect on IL-1α-induced IL-6 or IL-8 levels, co-stimulation inhibited the TGFβ1-induced increase in αSMA and blocked the gel contraction caused by TGFβ1. Combining IL-1α and TGFβ1 had no apparent effect on their canonical signaling pathways. In conclusion, IL-1α and TGFβ1 act synergistically to stimulate MMP3 expression in CF. Moreover, IL-1α has a dominant inhibitory effect on the phenotypic switch of CF to CMF induced by TGFβ1.     

Comparative proteomic analysis of cell cycle-dependent apoptosis induced by transforming growth factor-β

Transforming growth factor-β (TGF-β) can induce G2/M phase-dependent apoptosis and G1/S phase-dependent epithelial–mesenchymal transition (EMT) in hepatocytes, but the underlying mechanism remains poorly understood. In this study, we investigated alterations in the global proteome using two dimensional gel electrophoresis of AML-12 murine hepatocyte cells after treatment with TGF-β at several time points after synchronization in the G2/M or G1/S phase. Upon TGF-β treatment, the expression levels of 44 proteins were found to be significantly changed in cells synchronized in the G2/M phase. These proteins were identified by MALDI-TOF/TOF and classified into seven categories according to function. In addition, TGF-β induced downregulation of glutamine synthetase in cells in G2/M but not G1/S phase, and this was further confirmed by immunoblotting. Moreover, exogenous glutamine completely blocked TGF-β-induced apoptosis in G2/M and non-synchronized cells, whereas it had no effect on EMT, suggesting that the downregulation of glutamine synthetase is involved in G2/M phase-dependent apoptosis. These results provide new insight into the mechanism of the multifunctional effects of TGF-β and how apoptosis and EMT are regulated in the same type of cells.     

Effects of environmental conditions on cell growth and poly-β-hydroxybutyrate accumulation in Alcaligenes eutrophus

Influences of the control of glucose and oxygen concentrations on cell growth and poly--hydroxybutyrate (PHB) accumulation in Alcaligenes eutrophus were studied. Glucose affects both biosynthesis and glycolysis directly and the other pathways indirectly. PHB accumulation could also be stimulated under oxygen limitation conditions, but the final PHB content within the cells was less than in the case of nitrogen limitation. When the culture was shifted from the PHB accumulation state to balanced growth conditions, PHB degradation occurred in the cells. The cell growth was inhibited by high PHB content within the cells.     

A possible role of vimentin on the cell surface for the activation of latent transforming growth factor-β

Latent TGF-β (LTGF-β) has to be converted to active TGF-β for its activities. Previously, we reported that certain fragments of latency associated peptide (LAP) augmented LTGF-β activation via increase in binding of LTGF-β to the endothelial cell (EC) surface followed by cell-associated proteolysis. By searching for EC membrane proteins crosslinked with the LAP fragment, we identified the molecule bound to LAP fragment as vimentin. Moreover, the LAP fragment-induced LTGF-β activation was attenuated by anti-vimentin antibody. These results indicate that binding of the LAP fragment to vimentin on the cell surface is indispensable for LTGF-β activation by the LAP fragment.

Structured summary

MINT-6806227:vimentin (uniprotkb:P48616) binds (MI:0407) to LAP (uniprotkb:P18341) by competition binding (MI:0405)MINT-6806183:LAP (uniprotkb:P18341) binds (MI:0407) to vimentin (uniprotkb:P48616) by cross-linking studies (MI:0030)     

Thrombospondin-1-induced smooth muscle cell chemotaxis and proliferation are dependent on transforming growth factor-β2 and hyaluronic acid synthase

Angioplasty causes local vascular injury, leading to the release of thrombospondin-1 (TSP-1), which stimulates vascular smooth muscle cell (VSMC) migration and proliferation, important steps in the development of intimal hyperplasia. Transforming growth factor beta 2 (TGF-β2) and hyaluronic acid synthase (HAS) are two pro-stenotic genes upregulated in VSMCs by TSP-1. We hypothesized that inhibition of TGF-β2 or HAS would inhibit TSP-1-induced VSMC migration, proliferation, and TSP-1 signaling. Our data demonstrate that Inhibition of either TGF-β2 or HAS inhibited TSP-1-induced VSMC migration and proliferation. Activation of ERK 1 was decreased by TGF-β2 inhibition and unaffected by HAS inhibition. TGF-β2 and HAS are not implicated in TSP-1-induced thbs1 expression, while they are each implicated in TSP-1-induced expression of their own gene. In summary, TSP-1-induced VSMC migration and proliferation rely on intact TGF-β2 signaling and HAS function. TSP-1 activation of ERK 1 is dependent on TGF-β2. These data further expand our understanding of the complexity of TSP-1 cellular signaling and the involvement of TGF-β2 and HAS.     

Growth hormone induces transforming growth factor-β1 in podocytes: Implications in podocytopathy and proteinuria

Pituitary growth hormone (GH) is essential for growth, metabolism, and renal function. Overactive GH signaling is associated with impaired kidney function. Glomerular podocytes, a key kidney cell type, play an indispensable role in the renal filtration and express GH receptors (GHR), suggesting the direct action of GH on these cells. However, the precise mechanism and the downstream signaling events by which GH leads to diabetic nephropathy remain to be elucidated. Here we performed proteome analysis of the condition media from human podocytes and confirmed that GH-induces TGF-β1. Inhibition of GH/GHR stimulated-JAK2 signaling abrogates GH-induced TGF-β1 secretion. Mice administered with GH showed glomerular manifestations concomitant with proteinuria. Pharmacological inhibition of TGF-βR1 in mice prevented GH-induced TGF-β dependent SMAD signaling and proteinuria. Conditional deletion of GHR in podocytes protected mice from streptozotocin-induced diabetic nephropathy. GH and TGF-β1 signaling components expression was elevated in the kidneys of human diabetic nephropathy patients. Our study identifies that GH induces TGF-β1 in podocytes, contributing to diabetic nephropathy.     

Inhibitory effects of glycosaminoglycans on basal and stimulated transforming growth factor-β1 expression in mesangial cells: biochemical and structural considerations

A number of glycosaminoglycan (GAG) species related to heparin, dermatan sulfate (DeS) and chondroitin sulfate were tested for their ability to interfere with the physiological expression and/or pathological overexpression of the TGF-β1 gene. The influence of the molecular weight, molecular weight distribution, degree of sulfation and location of the sulfate groups was examined in an attempt to unveil fine relationships between structure and activity. The nature of the polysaccharide plays a major part, heparins proving able to inhibit both basal and stimulated TGF-β1 gene expression, DeSs being essentially inactive and chondroitin sulfates only inhibiting stimulated TGF-β1 gene expression. Within this frame, the particular physical and chemical properties of some GAGs appear to further modulate TGF-β1 gene response. Judging from our investigation, chondroitin sulfates seem the most promising for potential pharmacological applications in disorders characterized by fibrogenic TGF-β1 overexpression.     

LncRNA PAPAS promotes oral squamous cell carcinoma by upregulating transforming growth factor-β1

PAPAS is a recently identified long noncoding RNA (lncRNA) with inhibitory effects on ribosomal RNA synthesis. We studied the role of PAPAS in oral squamous cell carcinoma (OSCC). In the present study we showed that plasma PAPAS and transforming growth factor β1 (TGF-β1) were both upregulated in patients with OSCC, and were positively correlated only in patients with OSCC. Plasma levels of PAPAS were not significantly affected by AJCC stages and upregulation of PAPAS distinguished stage I OSCC patients from healthy controls. High plasma levels of PAPAS were followed by low overall survival rate. PAPAS overexpression led to upregulation of TGF-β1 in OSCC cells, while TGF-β1 treatment failed to significantly affect PAPAS. PAPAS overexpression and exogenous TGF-β1 treatment led to promoted invasion and migration of OSCC cells. In addition, TGF-β inhibitor attenuated the effects of PAPAS overexpression. Therefore, lncRNA PAPAS may promote OSCC by upregulating TGF-β1.