• Asian Pacific Journal of Cancer Prevention
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• v.13 no.11
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• pp.5709-5714
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• 2012

Objective: To evaluate the effects of curcumin on matrixmetalloproteinase-9 (MMP-9) and invasion ability induced by transforming growth factor-${\beta}1$ (TGF-${\beta}1$) in MDA-MB-231 cells and potential mechanisms. Methods: Human breast cancer MDA-MB-231 cells were used with the CCK-8 assay to measure the cytotoxicity of curcumin. After treatment with 10 ng/ml TGF-${\beta}1$, with or without curcumin (${\leq}10{\mu}M$), cell invasion was checked by transwell chamber. The effects of curcumin on TGF-${\beta}1$-stimulated MMP-9 and phosphorylation of Smad2, extracellular-regulated kinase (ERK), and p38 mitogen activated protein kinases (p38MAPK) were examined by Western blotting. Supernatant liquid were collected to analyze the activity of MMP-9 via zymography. Following treatment with PD98059, a specific inhibitor of ERK, and SB203580, a specific inhibitor of p38MAPK, Western blotting and zymography were employed to examine MMP-9 expression and activity, respectively. Results: Low dose curcumin (${\leq}10{\mu}M$) did not show any obvious toxicity to the cells, while $0{\sim}10{\mu}mol/L$ caused a concentration-dependent reduction in cell invasion provoked by TGF-${\beta}1$. Curcumin also markedly inhibited TGF-${\beta}1$-regulated MMP-9 and activation of Smad2, ERK1/2 and p38 in a dose- and time-dependent manner. Additionally, PD98059, but not SB203580, showed a similar pattern of inhibition of MMP-9 expression. Conclusion: Curcumin inhibited TGF-${\beta}1$-stimulated MMP-9 and the invasive phenotype in MDA-MB-231 cells, possibly associated with TGF-${\beta}$/Smad and TGF-${\beta}$/ERK signaling.

• Journal of Periodontal and Implant Science
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• v.25 no.1
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• pp.133-145
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• 1995

The migration and proliferation of periodontal ligament cells are desired goal of periodontal regeneration therapy. PDGF and $TGF-{\beta}1$ are well known to regulate the cell activity of mesenchymal origin cell. The purpose of this study was to determine the effects of these growth factors on human gingival fibroblast and periodontal ligament cell actvity, and to identify the regulatory effect of $TGF-{\beta}1$ on the response to PDGF by MIT assay. Human gingival fibroblast and periodontal ligament cells were cultured from extracted teeth for non-periodontal reason. Cultured human gingival fibroblast and periodontal ligament cells in vitro were treated with polyperpetide growth factor PDGF and $TGF-{\beta}1$ in both a dose and time - dependent manner. Cell morphology were determined by inverted microscope and cell acitivity were determined by MIT assay. The result of this study demonstrated that PDGF and $TGF-{\beta}1$ were not changed the morphology of these cell compared with control group. PDGF or $TGF-{\beta}1$ increased cell activity of periodontal ligament cell in dose and time dependent manner but gingival fibroblast were decreased to the level of control group at third day. Additionally, incubation with $TGF-{\beta}1$ addition to PDGF resulted in a enhanced cell activity of PDGF. Therefore, cell acitivty of gingival fibroblast were not changed compared with control group. This stiudy demonstrates that PDGF and $TGF-{\beta}1$ are major mitogens for human periodontal ligament cell in vitro, and $TGF-{\beta}1$ is a regulator of cell activity to PDGF in human gingival fibroblast and periodontal ligament cell.

• IMMUNE NETWORK
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• v.9 no.1
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• pp.27-33
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• 2009

Macrophages generated in vitro using macrophage-colony stimulating factor (M-CSF) and interleukin (IL)-6 from bone marrow cells (BM-Mp) are defective in antigen presenting cell (APC) function as shown by their ability to induce the proliferation of anti-CD3 mAb-primed syngeneic T cells. However, they do express major histocompatibility (MHC) class I and II molecules. accessory molecules and intracellular adhesion molecules. Here we demonstrate that the defective APC function of macrophages is mainly due to production of $TGF-{\beta}1$ by BM-Mp. Methods: Microarray analysis showed that $TGF-{\beta}1$ was highly expressed in BM-Mp, compared to a macrophage cell line, B6D. which exerted efficient APC function. Production of $TGF-{\beta}1$ by BM-Mp was confirmed by neutralization experiments of $TGF-{\beta}1$ as well as by real time-polymerase chain reaction (PCR). Results: Addition of $anti-TGF-{\beta}1$ monoclonal antibody to cultures of BM-Mp and anti-CD3 mAb-primed syngeneic T cells efficiently induced the proliferation of syngeneic T cells. Conversely, the APC function of B6D cells was almost completely suppressed by addition of $TGF-{\beta}1$. Quantitative real time-PCR analysis also confirmed the enhanced expression of $TGF-{\beta}1$ in BM-Mp. Conclusion: The defective APC function of macrophages generated in vitro with M-CSF and IL-6 was mainly due to the production of $TGF-{\beta}1$ by macrophages.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.20 no.2
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• pp.173-183
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• 1998

$TGF-{\beta}$ is one of growth factors that may be involved in the formation of bone and cartilage. Multiple studies demonstrate that $TGF-{\beta}$ is involved in regulating cell proliferation, differentiation and matrix synthesis, events observed in frature healing. The apperance of $TGF-{\beta}$ in the fracture during healing was evaluated by immunohistologic localization of $TGF-{\beta}$ using antibody. Twenty Sprauge-Dawley strain white male rats, each weiging about 150grams were used and divided two groups. The one group, the $2{\times}2mm$ bony defect was formed in the right femur. The other group, $4{\times}2mm$ bony defect was formed in right femur. Both group were sacrificed at 3day, 1, 2, 3, 4 week and femur were harvested, paraffin sections were stained with H & E, MT stain, immunihistochemical staining with $TGF-{\beta}$ antibody and observed under light microscope. The result were as follows: 1. New bone formation and cartilagenous tissue was seen at 3day. And in the $2{\times}2mm$ bony defect group, $TGF-{\beta}$ stained the cell surounding new bone. 2. The osteoclast and trabecular was seen at 1week. $TGF-{\beta}$ stained the osteoblast and in the $2{\times}2mm$ bony defect group was stained more than $4{\times}2mm$ bony defect group. 3. The lamella bone and trabecule was seen from 3, 4week, and $TGF-{\beta}$ stained almost negative. From the above finding, we could concluded that $TGF-{\beta}$ stained the osteoblast at early stage and 1week, the peak stain was seen from 1week, and then decreased, almost negative stain was seen at 3, 4week.

• Journal of Korean Neurosurgical Society
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• v.43 no.3
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• pp.149-154
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• 2008

Objective: The aim of this study is to elucidate the effects of transforming growth factor-${\beta}$ (TGF-${\beta}$)1 and L-ascorbic acid on proteoglycan synthesis, and the relationship between Sox9, proteoglycan, and TGF-${\beta}1$ in intervertebral disc cells. Methods: Human intervertebral disc tissue was sequentially digested to 0.2% pronase and 0.025% collagenase in DMEM/F-12 media and extracted cells were cultured in $37^{\circ}C$, 5% $CO_2$ incubator. When intervertebral disc cells were cultured with TGF-${\beta}1$ or L-ascorbic acid, the production level of sulfated glycosaminoglycan (sGAG) was estimated by dimethyl methyleneblue (DMMB) assay. The changes of Sox9 mRNA and protein levels via TGF-${\beta}1$ were detected by RT-PCR and Western blot analysis in each. Results: The amount of sGAG was increased with the lapse of time during incubation, and sGAG content of pellet cultured cells was much larger than monolayer culture. When primary cultured intervertebral disc cells in monolayer and pellet cultures were treated by TGF-${\beta}1$ 20 ng, sGAG content of experimental group was increased significantly compared to control group in both cultures. L-Ascorbic acid of serial concentrations (50-300 ug/ml) increased sGAG content of mono layer cultured intervertebral disc cells significantly in statistics. The co-treatment of TGF-${\beta}1$ and L-ascorbic acid increased more sGAG production than respective treatment. After treating with TGF-${\beta}1$, Sox9 mRNA and protein expression rates were significantly increased in disc cells compared with the control group. Conclusion: This study suggests that TGF-${\beta}1$ would increase sulfated glycosaminoglycan (sGAG) and other proteoglycans such as versican by elevating Sox9 mRNA and protein expressions in order.

• The Journal of Korean Medicine
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• v.22 no.3
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• pp.141-155
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• 2001

Objective : The aim of this study is to investigate the effect of Injin fractions on hepatic fibrosis induced by $TGF-{\beta}1$. Method : $TGF-{\beta}1$ mRNA, protein, $TGF-{\beta}1$ receptor, Smad family and PAI-I mRNA were studied in HepG2 cell, and the proliferation, connective tissue growth factor, fibronectin and collagen type I mRNA in T3891 fibroblast by quantitative RT-PCR, ELISA and thymidine incorporation assay. Results : On $TGF-{\beta}1$ mRNA and protein synthesis in HepG2, $H_2O$, butanol and hexane fractions of Injin showed inhibitory effect in a dose-dependent way. In the study on $TGF-{\beta}1$ receptor, Smad family and PAI-1 mRNA in HepG2, $H_2O$, butanol and hexane fraction of Injin showed inhibitory effect on the expression of PAI-1 in a dose-dependent way. On the proliferation of T3891 fibroblast induced by $TGF-{\beta}1$, $H_2O$, ethylacetate and butanol fractions of Injin showed inhibitory effect. In the study on the factors affected by $TGF-{\beta}1$, $H_2O$, ethylacetate and butanol fractions of Injin showed inhibitory effect on CTGF, and $H_2O$, butanol, chloroform and hexane fractions showed inhibitory effect on the expression of collagen type I, whereas no fraction showed inhibitory effect on the expression of fibronectin Conclusion : These results show that each fraction of Injin acts as a fibrosis inhibitory factor by itself or in combination, ultimately inhibiting liver cirrhosis.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1995.04a
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• pp.79-79
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• 1995

Although synthetic antisense oligodeoxynucleotides (ODNs) have been used to dissect gene function in vitro, technical difficulties of targeted delivery prevented the use of this approach for investigating the effect of gene products in vivo. Here we report the use of local delivery of antisense transforming growth factor-${\beta}$l (TGF-${\beta}$1) oligonucleotides to decrease the fibrosis in the skin wound. Adult wounds heal with scar-tissue formation, whereas fetal wounds heal without scarring and with a lesser inflammatory and cytokine response. We reasoned that strategy emptying antisense TGF-${\beta}$1 ODNs complementary to TGF-${\beta}$1 mRNA might decrease the scarring in dermal wound of mouse. To evaluate this concept, we tested the effects of antisense ODNs targeted to TGF-${\beta}$1 mRNA by topical application of the chemical on the skin wound. Phosphorothioate antisense ODNs was employed to retard their degradation. When antisense TGF-${\beta}$1 ODNs were applied into the wound site, there was a maked reduction of scar compared with control wound site, These effects of antisense TGF-${\beta}$1 ODNs on the scar formation were associated with decreased expression of TGF-${\beta}$1 gene. However sense TGF-${\beta}$l ODNs had no effect on expression of TGF-${\beta}$1 gene. Also, control wounds healed with excessive fibrosis, whereas the antisense treated wounds healed with less fibrosis. In conclusion, our results indicate that antisense TGF-${\beta}$1 ODNs could be used for amelioating scar formation during wound healing.

• Molecules and Cells
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• v.38 no.1
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• pp.20-25
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• 2015

TGF-${\beta}$ regulates pleiotropic cellular responses including cell growth, differentiation, migration, apoptosis, extracellular matrix production, and many other biological processes. Although non-Smad signaling pathways are being increasingly reported to play many roles in TGF-${\beta}$-mediated biological processes, Smads, especially receptor-regulated Smads (R-Smads), still play a central mediatory role in TGF-${\beta}$ signaling for epithelial-mesenchymal transition. Thus, the biological activities of R-Smads are tightly regulated at multiple points. Inhibitory Smad (I-Smad also called Smad7) acts as a critical endogenous negative feedback regulator of Smad-signaling pathways by inhibiting R-Smad phosphorylation and by inducing activated type I TGF-${\beta}$ receptor degradation. Roles played by Smad7 in health and disease are being increasingly reported, but the molecular mechanisms that regulate Smad7 are not well understood. In this study, we show that E3 ubiquitin ligase Itch acts as a positive regulator of TGF-${\beta}$ signaling and of subsequent EMT-related gene expression. Interestingly, the Itch-mediated positive regulation of TGF-${\beta}$ signaling was found to be dependent on Smad7 ubiquitination and its subsequent degradation. Further study revealed Itch acts as an E3 ubiquitin ligase for Smad7 polyubiquitination, and thus, that Itch is an important regulator of Smad7 activity and a positive regulator of TGF-${\beta}$ signaling and of TGF-${\beta}$-mediated biological processes. Accordingly, the study uncovers a novel regulatory mechanism whereby Smad7 is controlled by Itch.

• BMB Reports
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• v.55 no.8
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• pp.401-406
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• 2022

Ahnak, a large protein first identified as an inhibitor of TGF-β signaling in human neuroblastoma, was recently shown to promote TGF-β in some cancers. The TGF-β signaling pathway regulates cell growth, various biological functions, and cancer growth and metastasis. In this study, we used Ahnak knockout (KO) mice that underwent a 70% partial hepatectomy (PH) to investigate the function of Ahnak in TGF-β signaling during liver regeneration. At the indicated time points after PH, we analyzed the mRNA and protein expression of the TGF -β/Smad signaling pathway and cell cycle-related factors, evaluated the cell cycle through proliferating cell nuclear antigen (PCNA) immunostaining, analyzed the mitotic index by hematoxylin and eosin staining. We also measured the ratio of liver tissue weight to body weight. Activation of TGF-β signaling was confirmed by analyzing the levels of phospho-Smad 2 and 3 in the liver at the indicated time points after PH and was lower in Ahnak KO mice than in WT mice. The expression levels of cyclin B1, D1, and E1; proteins in the Rb/E2F transcriptional pathway, which regulates the cell cycle; and the numbers of PCNA-positive cells were increased in Ahnak KO mice and showed tendencies opposite that of TGF-β expression. During postoperative regeneration, the liver weight to body weight ratio tended to increase faster in Ahnak KO mice. However, 7 days after PH, both groups of mice showed similar rates of regeneration, following which their active regeneration stopped. Analysis of hepatocytes undergoing mitosis showed that there were more mitotic cells in Ahnak KO mice, consistent with the weight ratio. Our findings suggest that Ahnak enhances TGF-β signaling during postoperative liver regeneration, resulting in cell cycle disruption; this highlights a novel role of Ahnak in liver regeneration. These results provide new insight into liver regeneration and potential treatment targets for liver diseases that require surgical treatment.

• Tuberculosis and Respiratory Diseases
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• v.58 no.3
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• pp.267-276
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• 2005

Background : The transforming growth $factor-{\beta}1$ ($TGF-{\beta}1$) plays a key role in lung fibrosis. However, the molecular mechanisms involved in $TGF-{\beta}1$-induced lung fibrosis are unclear. $TGF-{\beta}1$ is the key inducer of myofibroblast transdifferentiation via de novo synthesis of ${\alpha}-smooth$ muscle actin (${\alpha}-SMA$). Since $TGF-{\beta}1$ signals through reactive oxygen species (ROS) and ROS have been shown to induce accumulation of extracellular matrix (ECM) in various tissues, this study examined if ROS play a role in $TGF-{\beta}1$-induced fibronectin secretion and ${\alpha}-SMA$ expression in human lung fibroblasts, MRC-5 cells. Methods : Growth arrested and synchronized MRC-5 cells were stimulated with $TGF-{\beta}1$ (0.2-10 ng/ml) in the presence or absence of N-acetylcysteine (NAC) or diphenyleneiodonium (DPI) for up to 96 hours. Dichlorofluorescein (DCF)-sensitive cellular ROS were measured by FACScan and secreted fibronectin and cellular ${\alpha}-SMA$ by Western blot analysis. Results : $TGF-{\beta}1$ increased the level of fibronectin secretion and ${\alpha}-SMA$ expression in MRC-5 cells in a dosedependent manner. Both NAC (20 and 30 mM) and DPI (1 and $5{\mu}M$) significantly inhibited $TGF-{\beta}1$-induced fibronectin and ${\alpha}-SMA$ upregulation. The $TGF-{\beta}1$-induced cellular ROS level was also significantly reduced by NAC and DPI. Conclusions : The results suggest that NADPH oxidase-dependent ROS play an important role in $TGF-{\beta}1$-induced fibronectin secretion and ${\alpha}-SMA$ expression in MRC-5 cells, which leads to myofibroblast transdifferentiation and progressive lung fibrosis.