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.     

Inhibin and activin modulate transforming growth factor-β-induced immunosuppression

Inhibin, activin, and transforming growth factor (TGF) inhibited lipopoly-saccharide (LPS)-induced lymphocyte proliferation in a dose-dependent fashion. These induced suppressions were neutralized by coincubation of a preparation of antibodies to inhibin and TGF, respectively. Inhibin and activin also facilitated TGF-mediated immunosuppression of LPS-induced proliferation of splenocytes. These gonadal proteins showed no effect on phytohemagglutinin-or concanavalin A (Con-A)-induced proliferation of lymphocytes. However, inhibin facilitated and activin inhibited the TGF-mediated immunosuppression in thymocytes stimulated by Con-A. These findings suggest that inhibin or activin by itself, and/or together with TGF, may play an important role in immune response.     

Effects of transforming growth factor-β1 and activin-A on in vitro porcine granulosa cell steroidogenesis

The mammalian ovarian cycle is a strictly regulated process that is dependent on the intimate interactions among the 3 cell types in the follicle — theca, granulosa, and oocyte. The cycle has been shown to be controlled by gonadotropins as well as locally produced peptide factors. In this study, an in vitro culture system was used to study the roles of 2 locally produced ovarian peptide factors, transforming growth factor-β₁ (TGF-β₁) and activin-A, on porcine granulosa cell steroidogenesis. Gonadotropin stimulated cultured porcine granulosa cells (from medium-sized follicles) were pretreated with 100 ng/ml follicle-stimulating hormone (FSH) for 48 h and then treated with 1 ng/ml TGF-β₁, 100 ng/ml activin-A, TGF-β₁ plus activin-A, or received no treatment (control) for 48 h, From our previous studies, the concentrations of the 2 growth factors were determined to produce maximal antisteroidogenic effects in porcine granulosa cells. Progesterone (P₄) production, estradiol-17β (E₂) production, and aromatase activity for gonadotropin-stimulated porcine granulosa cells treated with TGF-β₁, activin-A, and TGF-β₁ plus activin-A were significantly (P < 0.05) reduced fromthat of the control. The same procedures were conducted on basal steroidogenesis studies in which no pretreatment with FSH was performed. Both P₄ and E₂ production and aromatase activity for porcine granulosa cells treated with TGF-β₁, activin-A and TGF-β₁ plus activin-A were significantly (P < 0.05) inhibited compared with the control. Our results indicate that both TGF-β₁ and activin-A can inhibit FSH-stimulated and basal steroidogeneses in porcine granulosa cells and, thus, may act as local atretic factors during follicular development. When the 2 growth factors were given in combination at concentrations that would produce maximal steroidogenic inhibition, they were not able to produce a synergistic effect. These results are consistent with the current theory that TGF-β₁ and activin-A may act via the same messenger system, a serine-threonine kinase.     

Effect of β-alanyl-L-histidinato zinc on protein components in osteoblastic MC3T3-El cells: Increase in osteocalcin,insulin-like growth factor-I and transforming growth factor-β

The effect of -alanyl-L-histidinato zinc (AHZ) on protein components in osteoblastic MC3T3-E1 cells was investigated. Cells were cultured for 3 days at 37°C in CO₂ incubator in plastic dishes containing -modified minimum essential medium supplemented with 10% fetal bovine serum. After the cultures, the medium was exchanged for that containing 0.1% bovine serum albumin plus various concentrations of AHZ or other reagents, and the cells were cultured further 3 or 6 days. The homgenate of cells was analyzed with SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The presence of AHZ (10^–7 to 10^–5 M) caused an appreciable increase of many protein components in cells. Especially, the 67 killo-dalton (kDa) and 44 kDa proteins which are the major components from control cells were clearly increased by the presence of AHZ. Furthermore, the concentrations of osteocalcin, insulin-like growth factor-I and transforming growth factor- in the culture medium secreted from osteoblastic cells were markedly increased by the presence of AHZ (10^–6 and 10^–5 M). The effect of AHZ was a greater than that of zinc sulfate (10^–6 and 10^–5 M). The present findings suggest that AHZ can increase many proteins which are involved in the stimulation of bone formation and cell proliferation in osteoblastic cells.     

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.     

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.     

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.     

The extracellular matrix and transforming growth factor-β1: Tale of a strained relationship

Physiological tissue repair aims at restoring the mechano-protective properties of the extracellular matrix. Consequently, redundant regulatory mechanisms are in place ensuring that tissue remodeling terminates once matrix homeostasis is re-established. If these mechanisms fail, stromal cells become continuously activated, accumulate excessive amounts of stiff matrix, and fibrosis develops. In this mini-review, I develop the hypothesis that the mechanical state of the extracellular matrix and the pro-fibrotic transforming growth factor (TGF)-β1 cooperate to regulate the remodeling activities of stromal cells. TGF-β1 is stored in the matrix as part of a large latent complex and can be activated by cell contractile force that is transmitted by integrins. Matrix straining and stiffening lower the threshold for TGF-β1 activation by increasing the mechanical resistance to cell pulling. Different elements of this mechanism can be pharmacologically targeted to interrupt the mechanical positive feedback loop of fibrosis, including specific integrins and matrix protein interactions.     

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.

     

The roles of transforming growth factor-β and Smad3 signaling in adipocyte differentiation and obesity

We aimed at elucidating the roles of transforming growth factor (TGF)-β and Smad3 signaling in adipocyte differentiation (adipogenesis) and in the pathogenesis of obesity. TGF-β/Smad3 signaling in white adipose tissue (WAT) was determined in genetically obese (ob/ob) mice. The effect of TGF-β on adipogenesis was evaluated in mouse embryonic fibroblasts (MEF) isolated both from WT controls and Smad3 KO mice by Oil red-O staining and gene expression analysis. Phenotypic analyses of high-fat diet (HFD)-induced obesity in Smad3 KO mice compared to WT controls were performed. TGF-β/Smad3 signaling was elevated in WAT from ob/ob mice compared to the controls. TGF-β significantly inhibited adipogenesis in MEF, but the inhibitory effects of TGF-β on adipogenesis were partially abolished in MEF from Smad3 KO mice. TGF-β inhibited adipogenesis independent from the Wnt and β-catenin pathway. Smad3 KO mice were protected against HFD-induced insulin resistance. The size of adipocytes from Smad3 KO mice on the HFD was significantly smaller compared to the controls. In conclusion, the TGF-β/Smad3 signaling pathway plays key roles not only in adipogenesis but also in development of insulin resistance.     

Wnt3a upregulates transforming growth factor-β-stimulated VEGF synthesis in osteoblasts

It is recognized that Wnt3a affects bone metabolism via the canonical Wnt/β-catenin signalling pathway. We have previously shown that transforming growth factor-β (TGF-β) stimulates the synthesis of vascular endothelial growth factor (VEGF) via p44/p42 mitogen-activated protein (MAP) kinase, stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) and p38 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of Wnt3a on TGF-β-stimulated VEGF synthesis in these cells. Wnt3a, which alone had little effect on the VEGF levels, significantly enhanced the TGF-β-stimulated VEGF release. Lithium chloride and SB216763, inhibitors of glycogen synthase kinase 3β, markedly amplified the TGF-β-stimulated VEGF release. Wnt3a failed to affect the TGF-β-induced phosphorylation of Smad2, p44/p42 MAP kinase, p38 MAP kinase or SAPK/JNK. Wnt3a and lithium chloride strengthened the VEGF mRNA expression induced by TGF-β. These results strongly suggest that Wnt3a upregulates VEGF synthesis stimulated by TGF-β via activation of the canonical pathway in osteoblasts.     

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)     

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.