Enhancement of Metastatic and Invasive Capacity of Gastric Cancer Cells by Transforming Growth Factor-β1

Transforming growth factor-β (TGF-β),a multifunctional cytokine,exerts contradictory rolesin different kinds of cells.A number of studies have revealed its involvement in the progression of many typesof tumors.To investigate the effect of TGF-β on gastric carcinoma,SGC7901,BGC823 and MKN28 (aTGF-β-resistant cell line) adenocarcinoma clones were used.After pretreatment in serum-free medium withor without 10 ng/ml TGF-β1,their experimental metastatic potential,chemotaxis,and invasive and adhesiveability were measured.Furthermore,zymography for gelatinase was processed.Liver colonies were alsomeasured 4 weeks after inoculation of SGC7901,BGC823 and MKN28 in Balb/c nude mice,and an increasein the number of surface liver metastases was seen in SGC7901 (from 11.0±3.0 to 53.3±3.3) and BGC823(from 9.3±2.5 to 60.0±2.8) groups,whereas there was no difference between MKN28 groups (from 35.2±3.8 to 38.5±2.7).In vitro experiments showed that TGF-β1 increased the adhesion capacity of SGC7901and BGC823 cells to immobilized reconstituted basement membrane/fibronectin matrices and promoted theirpenetration through reconstituted basement membrane barriers.Zymography demonstrated that enhancedinvasive potential was partly due to the increased type Ⅳ collagenolytic (gelatinolytic) activity,but there wasno difference in type Ⅳ collagenolytic activity and other biological behaviors between MKN28 groups.Theseresults suggested that TGF-β1 might modulate the metastatic potential of gastric cancer cells by promotingtheir ability to break down and penetrate basement membrane barriers and their adhesive and motile activities.We speculated that TGF-β1 might act as a progression-enhancing factor in gastric cancer.Therefore blockageof TGF-β or TGF-β signaling might prevent gastric cancer cells from invading and metastasizing.     

Transforming Growth Factor-β1 Upregulates the Tight Junction and P-glycoprotein of Brain Microvascular Endothelial Cells

1. The present study was aimed at elucidating effects of transforming growth factor-beta (TGF-beta) on blood-brain barrier (BBB) functions with mouse brain capillary endothelial (MBEC4) cells. 2. The permeability coefficients of sodium fluorescein and Evans blue albumin for MBEC4 cells and the cellular accumulation of rhodamine 123 in MBEC4 cells were dose-dependently decreased after a 12-h exposure to TGF-beta1 (0.01-10 ng/mL). 3. The present study demonstrates that TGF-beta lowers the endothelial permeability and enhances the functional activity of P-gp, suggesting that cellular constituents producing TGF-beta in the brain may keep the BBB functioning.     

The Expression of Nerve Growth Factor Receptor on Schwann Cells and the Effect of These Cells on Regeneration of Axons in Traumatically Injured Human Spinal Cord

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Effects of Bone Morphogenetic Protein-2 and Transforming Growth Factor-β1 on Gene Expression of Decorin and Biglycan by Cultured Osteoblastic Cells

The influence of bone morphogenetic protein-2 (BMP-2) and transforming growth factor (TGF-) on the expression of small proteoglycans, decorin and biglycan was investigated in a clonal rat osteoblastic cell line, ROS-C26 (C26) cells, which is a potential osteoblast precursor cell line and capable of differentiating into mature osteoblasts after treatment with recombinant BMP-2 (rhBMP-2). Following the culture of C26 cells for 3, 6, and 9 days in the presence or absence of rhBMP-2, alkaline phosphatase activity increased in the rhBMP-2 treated cells in direct proportion to their differentiation into more mature osteoblastic cells, whereas decorin mRNA decreased in the cells, when compared to control cells without rhBMP-2 treatment. These results were evident 6 days after treatment. However, rhBMP-2 treatment had no effect on biglycan mRNA expression in the cells. Subsequently, after removal of rhBMP-2 from the culture media, the cells were further cultured for 24h with graded concentrations of TGF-1 (0, 0.1, 1.0, 5.0, and 10ng/ml). TGF-1 decreased decorin mRNA expression in the cells dose dependently, but did not affect their biglycan mRNA expression. Furthermore, either removal of rhBMP-2 from the culture media or addition of TGF-1 significantly decreased alkaline phosphatase activity of rhBMP-2-induced cells. These results indicate that osteoblastic differentiation is accompanied by increased alkaline phosphatase activity and decreased expression of decorin mRNA, but continuous expression of biglycan mRNA. Both rhBMP-2 and TGF-1 inhibit decorin mRNA expression in osteoblasts at varying stages of differentiation, but their effects on biglycan mRNA expression and alkaline phosphatase are different.     

Transforming Growth Factor-β1 Modulates Myofibroblastic Phenotype of Rat Palatal Fibroblastsin Vitro

The effects of transforming growth factor-β1 (TGF-β1) on normal rat palatal fibroblastsin vitrowere investigated in the present study in order to unravel the precise mechanisms by which the phenotypic modulation of fibroblasts occurs during the scar formation process. TGF-β1 dramatically changed the morphology of normal palatal fibroblasts from polygonal into an elongated shape, which was very similar to that of fibroblasts derived from experimental immature scar tissue in rat palatal mucosa. This morphological transition was concomitant with an increase in the expression of α-smooth muscle (α-SM) actin protein, a marker for myofibroblasts, when determined by immunocytochemistry. An immunoblot study also revealed that α-SM actin expression in palatal fibroblasts became evident after 24 h of TGF-β1 treatment and increased time-dependently up to 72 h. Northern blot analysis showed that TGF-β1 stimulated endogenous TGF-β1 mRNA expression in palatal fibroblasts within 24 h. Neither epidermal growth factor nor basic fibroblast growth factor had any effect on either α-SM actin expression or TGF-β1 mRNA expression. Pretreatment of palatal fibroblasts with TGF-β1 significantly increased the contractile capacity in a three-dimensional collagen gel culture, even when the culture medium was deprived of TGF-β1 for 72 h of the experimental period. Moreover, the contractility of scar fibroblasts, which highly expressed α-SM actin protein and TGF-β1 mRNA, was significantly lowered by a neutralizing antibody to TGF-β1. These data strongly suggest that TGF-β1 is a potential inducer of phenotypic expression of myofibroblasts in palatal fibroblasts and that autoinduction of TGF-β1 mRNA expression may play an important role in the scar formation process in palatal mucosa.     

Transforming Growth Factor-β1 Increases the Adhesion of MDA-MB-231 Mammary Adenocarcinoma Cells to the Microvascular Subendothelium

The increase of tumor cell adhesion to the Subendothelium in the presence of TGF-β1 is thought to be mediated by two major events: an enrichment of extracellular matrix proteins secreted by endothelial cells and an increase of the integrins on the surface of tumor cells. In this study, we analyzed the effect of TGF-β1 on the adhesion of a mammary adenocarcinoma cell line (MDA-MB-231) to the matrix of human microvascular endothelial cells (HMEC-1). The adhesion of TGF-β1 -treated tumor cells to a non-treated matrix or to purified matrix proteins was enhanced, white no increase was observed when non-treated tumor cells were let to adhere to a matrix secreted by HMEC-1 in the presence of the cytokine. Thus, the increase of cell adhesion was due to the effect of TGF-β1 on tumor cells and not to the matrix enrichment induced by this cytokine. The hyper-adhesion was inhibited by the RGD peptide and EDTA indicating that integrins were involved. Integrin subunits concentrations (α5, αv and β1) on the surface of TGF-β1-treated tumor cells were not modified, while confocal microscopy showed a reorganization of β1 integrin subunits on the cell surface and in the cytoplasm resulting in actin fibers reorganization in the cytoskeleton. This indicates that the enhanced adhesion of TGF-β1-treated MDA-MB-231 cells to the subendothelium is due to a qualitative change of integrins.     

Dynamic Monitoring of Cellular Remodeling Induced by the Transforming Growth Factor-β1

The plasticity of differentiated adult cells could have a great therapeutic potential, but at the same time, it is characteristic of progression of serious pathological states such as cancer and fibrosis. In this study, we report on the application of a real-time noninvasive system for dynamic monitoring of cellular plasticity. Analysis of the cell impedance profile recorded as cell index using a real-time cell analyzer revealed its significant increase after the treatment of prostate epithelial cells with the transforming growth factor-β1. Changes in the cell index profile were paralleled with cytoskeleton rebuilding and induction of epithelial–mesenchymal transition and negatively correlated with cell proliferation. This novel application of such approach demonstrated a great potential of the impedance-based system for noninvasive and real-time monitoring of cellular fate.     

Transforming Growth Factor-β1 and -β2 in Gastric Precancer and Cancer and Roles in Tumor-Cell Interactions with Peripheral Blood Mononuclear Cells In Vitro

Transforming growth factor-β1 (TGF-β1) and -β2 are correlated with poorer prognosis in gastric cancer (GC), which act in both tumor and immune cells. However, their expressions in precancer and tumor-cell interactions with peripheral blood mononuclear cells (PBMCs) remain unclear. Protein levels of TGF-β1 and -β2 were analyzed by immunohistochemistry and corresponding mRNA levels were determined by quantitative real-time polymerase chain reaction in 93 surgical and biopsy specimens. Serum TGF-β concentration was detected by enzyme-linked immunosorbent assays. AGS and MKN45 cell lines were directly or indirectly cocultured with PBMCs in vitro. TGF-β and Smad molecules were detected after cocultures and the growths of GC cells and PBMCs were assessed by cell proliferation assay. The results showed positive staining for TGF-β1 was detected in 20% of control samples, 52.3% of precancer, 59.1% of early GC and 66.7% of advanced GC samples, correlated with lesion progression (χ² = 9.487, P = 0.002). All tissues were positive for TGF-β2. TGF-β1 mRNA levels were increased in advanced cancers, while TGF-β2 increased earlier. TGF-β1 mRNA levels were higher in tumor than in peritumor, which positively correlated with Smad2 and Smad7. Serum TGF-β levels were significantly higher in patients with early and advanced cancers compared to controls (TGF-β1∶50.08±4.38 and 45.76±5.00 vs. 27.78±6.11 ng/mL; TGF-β2∶133.61±21.90 and 111.34±15.76 vs. 59.41±15.42 ng/mL, both P<0.05). The levels of TGF-β1 mRNA and cytokine secretion were higher in GC cells after direct coculture compared to indirect culture. TGF-β1 was decreased and TGF-β2 was increased in PBMCs after cocultures. Moreover, TGF-β1 inhibited the viability of PBMCs but not cancer cells. Collectively, neoplastic transformation may be an early event involving the increase of TGF-β1 in the general and local environment. TGF-β1 production is promoted by the direct interaction between GC cells and PBMCs, which might facilitate cancer development.     

Pin1 Promotes Transforming Growth Factor-��-induced Migration and Invasion

Transforming growth factor-β (TGF-β) regulates a wide variety of biological activities. It induces potent growth-inhibitory responses in normal cells but promotes migration and invasion of cancer cells. Smads mediate the TGF-β responses. TGF-β binding to the cell surface receptors leads to the phosphorylation of Smad2/3 in their C terminus as well as in the proline-rich linker region. The serine/threonine phosphorylation sites in the linker region are followed by the proline residue. Pin1, a peptidyl-prolyl cis/trans isomerase, recognizes phosphorylated serine/threonine-proline motifs. Here we show that Smad2/3 interacts with Pin1 in a TGF-β-dependent manner. We further show that the phosphorylated threonine 179-proline motif in the Smad3 linker region is the major binding site for Pin1. Although epidermal growth factor also induces phosphorylation of threonine 179 and other residues in the Smad3 linker region the same as TGF-β, Pin1 is unable to bind to the epidermal growth factor-stimulated Smad3. Further analysis suggests that phosphorylation of Smad3 in the C terminus is necessary for the interaction with Pin1. Depletion of Pin1 by small hairpin RNA does not significantly affect TGF-β-induced growth-inhibitory responses and a number of TGF-β/Smad target genes analyzed. In contrast, knockdown of Pin1 in human PC3 prostate cancer cells strongly inhibited TGF-β-mediated migration and invasion. Accordingly, TGF-β induction of N-cadherin, which plays an important role in migration and invasion, is markedly reduced when Pin1 is depleted in PC3 cells. Because Pin1 is overexpressed in many cancers, our findings highlight the importance of Pin1 in TGF-β-induced migration and invasion of cancer cells.     

Hypoxia Induces Transforming Growth Factor-β1 Gene Expression in the Pulmonary Artery of Rats via Hypoxia-inducible Factor-1α

The present study was undertaken to investigate the dynamic expression of hypoxia induciblefactor-1 α (HIF-1α) and transforming growth factor-β1 (TGF-β1) in hypoxia-induced pulmonary hypertensionof rats.It was found that mean pulmonary arterial pressure (mPAP) increased significantly after 7 d ofhypoxia.Pulmonary artery remodeling index and right ventricular hypertrophy became evident after 14 d ofhypoxia.HIF-1α mRNA staining was less positive in the control,hypoxia for 3 d and hypoxia for 7 d,butbegan to enhance significantly after 14 d of hypoxia,then remained stable.Expression of HIF-1 α protein inthe control was less positive,but was up-regulated in pulmonary arterial tunica intima of all hypoxic rats.TGF-β1 mRNA expression in pulmonary arterial walls was increased significantly after 14 d of hypoxia, butshowed no obvious changes after 3 or 7 d of hypoxia.In pulmonary tunica adventitia and tunica media,TGF-β1 protein staining was less positive in control rats,but was markedly enhanced after 3 d of hypoxia,reaching its peak after 7 d of hypoxia,and then weakening after 14 and 21 d of hypoxia.Western blottingshowed that HIF- 1α protein levels increased significantly after 7 d of hypoxia and then remained at a highlevel. TGF-β1 protein level was markedly enhanced after 3 d of hypoxia,reaching its peak after 7 d ofhypoxia,and then decreasing after 14 and 21 d of hypoxia.Linear correlation analysis showed that HIF-1αmRNA, TGF-β1 mRNA, TGF-β1 protein were positively correlated with mPAP,vessel morphometry andright ventricular hypertrophy index.TGF-β1 protein (tunica adventitia) was negatively correlated withHIF-lα mRNA.Taken together,our results suggest that changes in HIF-lα and TGF-β1 expression afterhypoxia play an important role in hypoxia-induced pulmonary hypertension of rats.     

p38 Mitogen-Activated Protein Kinase Affects Transforming Growth Factor-β/Smad Signaling in Human Dental Pulp Cells

Transforming Growth Factor-β (TGF-β) plays an essential role in differentiation of dental pulp cells into odontoblasts during reparative dentine formation. However, the mechanism by which TGF-β stimulates dental repair remains rather obscure. Human dental pulp cells were used as an in vitro model in the present work. We showed that TGF-β signaled through mitogen-activated protein kinases (MAPKs), such as ERK1/2 and p38, along with Smad pathway. Distinct pathways exerted different time response. SB203580, a specific p38 MAPK inhibitor, reduced phosphorylation of Smad3, while it slightly enhanced phosphorylation of Smad2. Increased phosphorylation of ERK1/2 and p38 confirmed that SB203580 did not block activation of TGF-β receptors. In addition, the inhibition of ERK1/2 activity with MEK1/2 inhibitor U0126 increased TGF-β mediated phosphorylation of Smad3. Our results suggest that p38 affects the phosphorylation of Smad2 and Smad3 differentially during TGF-β signaling in human dental pulp cells and ERK1/2 might be involved in the process.     

Dual Function of Transforming Growth Factor-β in the Control of Proliferation and Apoptosis of Cells of the Nervous System

Transforming growth factor- (TGF-) is an agent that gave the name to an extensive superfamily of congeneric cytokines playing important roles in numerous physiological and pathological processes. TGF- is involved in a few signal pathways controlling growth, differentiation, and death (apoptosis) of the nerve cells. Yet, it was found that the role of TGF- in each of these processes is dual: it can act either as their stimulator or as an inhibitor. This review describes examples and principal mechanisms of the dual functions of TGF- in its regulatory influences realized in the mammalian nervous system.     

Transforming Growth Factor-�� Promotes Recruitment of Bone Marrow Cells and Bone Marrow-derived Mesenchymal Stem Cells through Stimulation of MCP-1 Production in Vascular Smooth Muscle Cells

Bone marrow-derived progenitor cells have recently been shown to be involved in the development of intimal hyperplasia after vascular injury. Transforming growth factor-β (TGF-β) has profound stimulatory effects on intimal hyperplasia, but it is unknown whether these effects involve progenitor cell recruitment. In this study we found that although TGF-β had no direct effect on progenitor cell recruitment, conditioned media derived from vascular smooth muscle cells (VSMC) stimulated with TGF-β induced migration of both total bone marrow (BM) cells and BM-mesenchymal stem cells (MSC) and also induced MSC differentiation into smooth muscle like cells. Furthermore, overexpression of the signaling molecule Smad3 in VSMC via adenovirus-mediated gene transfer (AdSmad3) enhanced the TGF-β''s chemotactic effect. Microarray analysis of VSMC stimulated by TGF-β/AdSmad3 revealed monocyte chemoattractant protein-1 (MCP-1) as a likely factor responsible for progenitor cell recruitment. We then demonstrated that TGF-β through Smad3 phosphorylation induced a robust expression of MCP-1 in VSMC. Recombinant MCP-1 mimicked the stimulatory effect of conditioned media on BM and MSC migration. In the rat carotid injury model, Smad3 overexpression significantly increased MCP-1 expression after vascular injury, consistent with our in vitro results. Interestingly, TGF-β/Smad3-induced MCP-1 was completely blocked by both Ro-32-0432 and rotterlin, suggesting protein kinase C-δ (PKCδ) may play a role in TGF-β/Smad3-induced MCP-1 expression. In summary, our data demonstrate that TGF-β, through Smad3 and PKCδ, stimulates VSMC production of MCP-1, which is a chemoattractant for bone marrow-derived cells, specifically MSC. Manipulation of this signaling system may provide a novel approach to inhibition of intimal hyperplasia.