Different Concentrations Of TGF-β1 Research Articles

Chitosan-based endodontic irrigation solutions and TGF-β1 treatment: Creating the most favourable environment for the survival and proliferation of stem cells of the apical papilla in vitro.

The dental pulp's environment is essential for the regulation of mesenchymal stem cells' homeostasis and thus, it is of great importance to evaluate the materials used in regenerative procedures. To assess invitro (i) the effect of chitosan nanoparticles, 0.2% chitosan irrigation solution, Dual Rinse®, 17% EDTA, 10% citric acid and 2.5% NaOCl on DSCS viability; (ii) the effect of different concentrations of TGF-β1 on DCSC proliferation; and (iii) whether treatment with TGF-β1 following exposure to the different irrigation solutions could compensate for their negative effects. (i) DSCS were treated with three dilutions (1:10, 1:100 and 1:1000) of the six irrigation solutions prepared in DMEM for 10 and 60 min to assess the effect on viability. (ii) The effect of different concentrations (0, 1, 5 and 10 ng/mL) of TGF-β1 on DCSC proliferation was assessed at 1, 3 and 7 days. (iii) The proliferative effect of TGF-β1 following 10-min exposure to 1:10 dilution of each irrigation solution was also tested. We used MTT assay to assess viability and proliferation. We performed statistical analysis using Prism software. (i) The different endodontic irrigation solutions tested showed a significant effect on cell viability (p ≤ .0001). Significant interactions between the endodontic irrigation solutions and their dilutions were also found for all parameters (p ≤ .0001). Chitosan nanoparticles and 0.2% chitosan irrigation solution were the least cytotoxic to DSCS whilst 2.5% NaOCl was the most cytotoxic followed by 17% EDTA. (ii) TGF-β1 at concentrations of 1 and 5 ng/mL resulted in significantly higher proliferation compared to the control group. (iii) Exposure to 17% EDTA or 2.5% NaOCl for 10 min was sufficient to make DSCS cells refractory to the proliferative effects of TGF-β1. DSCS groups treated with TGF-β1 following exposure to chitosan nanoparticles, 0.2% chitosan irrigation solution, Dual Rinse® and 10% CA demonstrated significantly higher proliferation compared to non-TGF-β1-treated groups (p ≤ .0001, p ≤ .0001, p ≤ .0001 and p = .01 respectively). The current study offers data that can be implemented to improve the outcome of regenerative endodontic procedures by using less toxic irrigation solutions and adding TGF-β1 to the treatment protocol.

TGFβ1-Pretreated Exosomes of Wharton Jelly Mesenchymal Stem Cell as a Therapeutic Strategy for Improving Liver Fibrosis

Background: Mesenchymal stem cells (MSCs) are the most promising tools for cell treatment and human tissue regeneration, e.g., in liver fibrosis. Mesenchymal stem cells repair tissue damage through paracrine mediators such as exosomes. Types and concentrations of inflammatory mediators, including transforming growth factor-beta (TGFβ1), in MSCs microenvironment can affect MSCs’ function and therapeutic potency. Objectives: This experimental study aimed to explore the effects of Wharton jelly MSCs (WJ-MSCs) exosomes on fibrotic gene expression and Smad2/3 phosphorylation (phospho-Smad2/3 (p-Smad2/3)). Moreover, we further investigated whether WJ-MSCs pretreatment with different concentrations of TGFβ1 changes the anti-fibrotic properties of their exosomes. Methods: After isolation from the umbilical cord, WJ-MSCs were characterized by observing differentiation and measuring surface biomarkers using flowcytometry. The WJ-MSC-derived exosomes were extracted and identified using transmission electron microscopy (TEM), dynamic light scattering (DLS), and western blotting. Real-time PCR and western blot for extracellular matrix (ECM) and p-Smad2/3 expression detection were used to investigate the effect of exosomes from untreated and TGFβ1-pretreated WJ-MSCs on activated hepatic stellate cells (HSCs). Results: Phospho-Smad2/3, α-smooth muscle actin (α-SMA), and collagen1α1 levels were enhanced following treatment with TGFβ1, whereas E-cadherin was decreased. However, the outcomes were reversed after treatment with WJ-MSC-derived exosomes. Exosomes from TGFβ1-pretreated WJ-MSCs induced a significant decrease in p-Smad2/3 levels in activated HSCs, accompanied by the upregulation of E-cadherin gene expression and downregulation of α-SMA and collagen1α1 when compared to untreated WJ-MSC-derived exosomes. The p-Smad2/3 proteins were significantly decreased (fold change: 0.23, P-value < 0.0001) after exposure to low-dose TGFβ1-pretreated WJ-MSC-derived exosomes (0.1 ng/mL), showing the best effect on activated HSCs. Conclusions: Exosomes derived from untreated WJ-MSCs could regress TGFβ-Smad2/3 signaling and the expression of fibrotic markers in activated LX-2 cells. However, these effects were significantly profound with applying exosomes derived from 0.1 ng/mL TGFβ-pretreated WJ-MSCs. We also observed the dose-response effects of TGFβ on WJ-MSCs-derived exosomes. Therefore, exosomes derived from TGFβ-pretreated WJ-MSCs may be critical in improving fibrosis and benefit liver fibrosis patients.

Transforming growth factor-β1 negatively regulates SOCS7 via EGR1 during wound healing

BackgroundTGF-β1 promotes keratinocyte migration and re-epithelialization of cutaneous wounds during the wound healing process. Decreased SOCS7 expression has been associated with increased healing potential. However, the relationship between TGF-β1 and SOCS7 in wound re-epithelialization remains unclear.ObjectivesTo investigate the relationship between TGF-β1 and SOCS7 in the re-epithelialization of keratinocytes during skin wound healing.MethodsThe expression of SOCS7 under different concentrations of TGF-β1 was detected by WB and qPCR. The migration ability of keratinocytes was detected by scratch and Transwell assay. Protein interactions were detected by ChIP and luciferase assay. The effect of SOCS7 on skin healing in mice was detected in animal model.ResultsIn this study, we found that SOCS7 was downregulated by TGF-β1 and that overexpression of SOCS7 led to suppression of TGF-β1-induced keratinocyte migration through inhibition of the PI3K/AKT and MEK/ERK pathways. Also, TGF-β1 negatively regulated SOCS7 expression at the transcriptional level through the binding of EGR1 to the EGR1/SP1 overlapping binding sites in the SOCS7 promoter.ConclusionTaken together, our findings show that TGF-β1-induced EGR1 expression is required for repression of SOCS7, which promotes keratinocyte migration and re-epithelialization during wound healing. Finally, our study identifies the TGF-β1/EGR1/SOCS7 pathway as a potential therapeutic target to promote wound healing.5iNumoHWC7LtyUXVHhpfUVVideo

SREBP2 promotes the viability, proliferation, and migration and inhibits apoptosis in TGF-β1-induced airway smooth muscle cells by regulating TLR2/NF-κB/NFATc1/ABCA1 regulatory network

ABSTRACT Asthma is a respiratory disease with complex pathogenesis. Sterol-responsive element-binding proteins 2 (SREBP2) was found to bind to promoter sequences of ABCA1 to suppress ABCA1 promoter activity. This study aimed to explore the expression level of SREBP2 and ATP-binding cassette transporter A1 (ABCA1), and their effects on the development of airway smooth muscle cells (ASMCs) in asthma. ASMCs were treated with different concentrations of TGF-β1 (0, 0.5, 1, 5 and 10 ng/mL). Short hairpin SREBP2 (shSREBP2), SREBP2, shABCA1 or ABCA1 were transfected into ASMCs. Cell viability, proliferation, apoptosis, migration, and the expression of SREBP2, ABCA1 and related pathway proteins were detected by MTT assay, Brdu staining, flow cytometer, Transwell assay, qRT-PCR, and Western blotting, respectively. The results showed that TGF-β1 increased the viability, proliferation, migration and inhibited apoptosis in ASMCs. Moreover, TGF-β1 also decreased the expression of ABCA1, cleaved caspase-3, cleaved PARP, E-cadherin, and increased the expression of vimentin, TLR2, p-p65 and NFATc1. SREBP2 knockdown alleviated these TGF-β1-induced changes. SREBP2 overexpression inhibited ABCA1 expression and apoptosis, and promoted cell migration and the expression of TLR2, p-p65, NFATc1 in ASMCs. ABCA1 overexpression alleviated these SREBP2-induced promoting and inhibition effects. In conclusion, SREBP2 activates TLR2/NF-κB/NFATc1 regulatory network and promotes TGF-β1-induced cell movement through inhibiting ABCA1 expression.

Probing TGF-β1-induced cytoskeletal rearrangement by fluorescent-labeled silica nanoparticle uptake assay

Cytoskeletal proteins are essential in maintaining cell morphology, proliferation, and viability as well as internalizing molecules in phagocytic and non-phagocytic cells. Orderly aligned cytoskeletons are disturbed by a range of biological processes, such as the epithelial–mesenchymal transition, which is observed in cancer metastasis. Although many biological methods have been developed to detect cytoskeletal rearrangement, simple and quantitative in vitro approaches are still in great demand. Herein, we applied a flow cytometry-based nanoparticle uptake assay to measure the degree of cytoskeletal rearrangement induced by transforming growth factor β1 (TGF-β1). For the assay, silica nanoparticles, selected for their high biocompatibility, were fluorescent-labeled to facilitate quantification with flow cytometry. Human keratinocyte HaCaT cells were treated with different concentrations of TGF-β1 and then exposed to FITC-labeled silica nanoparticles. Increasing concentrations of TGF-β1 induced gradual changes in cytoskeletal rearrangement, as confirmed by conventional assays. The level of nanoparticle uptake increased by TGF-β1 treatment in a dose-dependent manner, indicating that our nanoparticle uptake assay can be used as a quick and non-destructive approach to measure cytoskeletal rearrangement.

The effects of TGF-β1 on staphylococcus epidermidis biofilm formation in a tree shrew biomaterial-centered infection model

BackgroundTransforming growth factor-β1 (TGF-β1) has a wide range of biological functions. It antagonizes lymphocyte response, inhibits pro-inflammatory cytokines, and serves as a signal to turn off the immune response and inflammatory response. To study the correlation between TGF-β1 and T helper (Th)1/Th2 cytokine levels in tree shrews, and to explore the effects of different levels of TGF-β1 on central venous catheter (CVC)-centered Staphylococcus epidermidis biofilm formation in tree shrews.MethodsTree shrews were injected with different concentrations of TGF-β1, and venous blood was drawn after 48 h to measure the levels of Th1 and Th2 cytokines. A CVC was placed into the femoral vein, and TGF-β1 at different concentrations and PIA− (ATCC12228) and PIA+ (ATCC35984) standard strains of Staphylococcus epidermidis were injected into the tree shrews to establish a biomaterial-centered infection (BCI) model. After 72 h, the CVC was removed, and biofilm formation was detected using the API bacterial identification system, semi-quantitative biofilm formation assay, and scanning electron microscopy.ResultsIn the groups treated with TGF-β1 at different concentrations, the levels of Th1 cytokines interleukin-2 (IL-2), tumor necrosis factor (TNF), and interferon-γ (IFN-γ) were lower than those of normal group, while the levels of Th2 cytokines IL-6, IL-4 and IL-10 were higher than those of normal group. In the TGF-β1 groups at different concentrations, the positive rate of Staphylococcus epidermidis ATCC35984 biofilm formation was higher than that in non-TGF-β1 group, while there was no significant difference in the positive rate of Staphylococcus epidermidis ATCC12228 biofilm formation compared with that of the non-TGF-β1 group.ConclusionsTGF-β1 causes the imbalance of Th1/Th2 cytokines and Th1/Th2 shift in tree shrews, leading to Th1 cell-led decline in cellular immune function. TGF-β1 promotes PIA+ Staphylococcus epidermidis biofilm formation in the tree shrew BCI model, but it has no significant influence on PIA-Staphylococcus epidermidis biofilm formation on the surface of CVCs.

The influences of TGF-β1 upon the human adenocarcinoma cell of lung A549 and cellular immunity.

BackgroundThe cellular immunity of lung cancer patients is mainly the immune response of T cells, which plays an important role in tumour cell killing and immune surveillance. Transforming growth factor 1 (TGF-β1) is secreted by tumour cells that can suppress the immune response and is an important group of immune down-regulation factors. Our study aims to investigate the effect of TGF-β1 on the morphology and cellular immune function of A549 and peripheral blood mononuclear cells (PBMCs).MethodsA549 cell line was cultured, PBMCs were cultured with different concentrations of TGF-β1, and the morphology of A549 cells and PBMCs were seen. The levels of interleukin (IL)-2, IL-4, IL-6, IL-10, IFN-γ, and TNF and the numbers of CD3, CD4, CD8, CD4/CD8, and CD3 CD25 and CD4 CD25 in PBMCs were detected.ResultsDuring co-culture of A549 with PBMCs, TGF-β1 can induced A549 showing epithelial-to-mesenchymal transition, enhanced its ability of migration and infiltration. Simultaneously, TGF-β1 can depressing the growth and proliferation of PBMCs, inhibiting T-cell activation, and accelerating the PBMCs apoptosis. TGF-β1 can inhibits A549 Th1 related-cytokines, enhance Th2 related-cytokines, cause the disorder of Th1/Th2, resulting in the Th1 cellular dominate immunity decline.ConclusionsTGF-β1 may affect the secretion of related cytokines, hinder the activation of T lymphocytes, destroy the immune surveillance and killing effect of the body, and thus inhibit the cellular immunity.

Parthenolide inhibits transforming growth factor β1-induced epithelial-mesenchymal transition in colorectal cancer cells.

Background/AimsTransforming growth factor-β1 (TGF-β1) induction of epithelial-mesenchymal transition (EMT) is one of the mechanisms by which colorectal cancer (CRC) cells acquire migratory and invasive capacities, and subsequently metastasize. Parthenolide (PT) expresses multiple anti-cancer and anti-inflammatory activities that inhibit nuclear factor κB by targeting the IκB kinase complex. In the present study, we aimed to investigate whether PT can inhibit TGF-β1-induced EMT in CRC cell lines.MethodsHT-29 and SW480 cell lines were used in the experiment. Cell viability was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and sub-G1 analysis was measured by flow cytometry. The induction of EMT by TGF-β1 and inhibition of the process by PT was analyzed by phase contrast microscopy, wounding healing, cellular migration and invasion assays, and Western blotting.ResultsTGF-β1 inhibits HT-29 cell proliferation, but has no effect on SW480 cell proliferation; different concentrations of TGF-β1 did not induce apoptosis in HT-29 and SW480 cells. PT attenuates TGF-β1-induced elongated, fibroblast-like shape changing in cells. PT inhibits TGF-β1-induced cell migration and cell invasion. In addition, other EMT markers such as β-catenin, Vimentin, Snail, and Slug were suppressed by PT, while E-cadherin was increased by PT.ConclusionsOur findings show that PT inhibits TGF-β1-induced EMT by suppressing the expression of the mesenchymal protein and increasing expression of the epithelial protein. These findings suggest a novel approach for CRC treatment by suppression of TGF-β1-induced EMT.

Tanshinone IIA Inhibits TGF-β1-Mediated Fibroblasts Proliferation by Down-Regulating lncRNA-HSRL/SNX9/p-Smad3 Signaling

Tanshinone IIA (TSIIA), one of the components of Salvia miltiorrhiza (Danshen), has been reported to inhibit proliferation in hypertrophic scars (HS). However, the molecular mechanisms underlying TSIIA regulating HS still remain largely unknown. In this study, we used different concentrations of TGF-β1 to simulate Human skin fibroblasts (HSFs) to mimic scar formation, followed by treatment with various concentrations of TSIIA. We observed TSIIA could effectively attenuate TGF-β1-mediated fibroblasts proliferation in a dose-dependent manner using CCK-8, Edu staining and colony formation assays. Meanwhile, TSIIA could effectively down-regulated the expression of α-SMA, VEGFA, Collagen 1, HSRL, SNX9 and p-Smad2/3 in TGF-β1-stimulated HSFs in a dose-dependent manner using quantitative real time PCR and western blotting analysis. Through loss-of-function and rescue experiments, we found SNX9 overexpression reversed the effects of HSRL knockdown on TGF-β1- stimulated HSFs. Mechanically, the interaction between HSRL and SNX9 was validated by RNA pull-down assay in HSFs. Using Co-IP assay, we confirmed the p-Smad3 that interaction with SNX9 was weakened after TSIIA treatment in TGF-β1-stimulated HSFs. In conclusions, these findings indicate that specifically targeting HSRL/SNX9/p-Smad3 might enhance the suppressive effects of TSIIA in HS pathogenesis. Funding Statement: Guangdong natural science project [2018A0303130234] and excellent young teachers training project in higher education of Guangdong [YQ2015099] which supported this experiment. Declaration of Interests: The authors declare no conflicts of interest. Ethics Approval Statement: Not required.

Inhibitory effect of transforming growth factor-β1 on expression of N-myc downstream regulatory gene 2 in human renal tubular epithelial cell line HK-2 and its significance

Objective To investigate the effect of transforming growth factor-β1 (TGF-β1) on the expression of N-myc downstream regulatory gene 2 (NDRG2) in human renal tubular epithelial cell line HK-2. Methods The control group and the experimental group were set up according to whether or not the TGF-β1 was incubated. Western blotting and real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) were applied to detect the expression of E-cadherin, α-smooth muscle actin (α-SMA), Vimentin and Snail protein in epithelial cells. The expression of NDRG2 in human renal tubular epithelial cells (HK-2) treated with different concentrations of TGF-β1 was detected by Western blotting and RT-qPCR. Results The results showed that the protein expression level expression of E-cadherin in the control group and the experimental group was (1.0±0.2) vs. (0.3±0.2) (t=4.850, P=0.008), that of α-SMA was (1.0±0.1) vs. (2.6±0.4) (t=6.721, P=0.003), that of Vimentin was (1.0±0.1) vs. (2.6±0.4) (t=6.721, P=0.003), and that of Snail was (1.0±0.1) vs. (2.5±0.2) (t=11.620, P=0.000). In the control group and the experimental group, the mRNA expression level of E-cadherin was (1.0±0.2) vs. (0.2±0.1) (t=7.686, P=0.002), that of α-SMA was (1.0±0.3) vs. (2.4±0.3) (t=5.715, P=0.005), that of Vimentin was (1.1±0.15) vs. (2.6±0.5) (t=4.977, P=0.008), and that of Snail was (0.9±0.2) vs. (1.9±0.2) (t=6.124, P=0.004). At the same time, after incubation with TGF-β1, NDRG2 in HK-2 cells was significantly down-regulated at both protein and mRNA levels. The levels of protein expression in each group were (1.00±0.04 vs. 0.80±0.03 vs. 0.50±0.05 vs. 0.30±0.03 vs. 0.20±0.02) (P=0.019), and mRNA expression in each group were (1.00±0.05 vs. 0.70±0.03 vs. 0.50±0.04 vs. 0.30±0.02 vs. 0.20±0.05) (P=0.021). Especially when incubated with TGF-β1 concentration greater than 10 ng/ml, the difference was statistically significant. Conclusion TGF-β1 can promote the process of EMT in HK-2 cells, and TGF-β1 can down-regulate the expression of NDRG2 in protein and RNA in HK-2 cells. Key words: N-myc downstream regulatory gene 2; Transforming growth factor-β1; Renal tubular epithelial cells; Epithelial-mesenchymal transition

TGF-β1 stimulates epithelial-mesenchymal transition mediated by ADAM33.

The present study aimed to determine the effects of transforming growth factor (TGF)-β1 on disintegrin and metalloproteinase domain-containing protein 33 (ADAM33) expression in airway epithelial cells in order to investigate the association between ADAM33 expression and TGF-β1-induced epithelial to mesenchymal transition (EMT), and to further explore the mechanisms underlying the role of ADAM33 in airway remodeling in asthma. The human bronchial epithelial cell line HBE was transfected with small interfering RNA targeting ADAM33 (siADAM33) and treated with different concentrations of TGF-β1 (10, 20 or 30 ng/ml), while untransfected cells were used as controls. At 72 h after treatment, cellular morphology and immunohistochemical staining were observed under a microscope. The protein and mRNA expression levels of ADAM33 and the EMT markers E-cadherin and vimentin were detected by western blot analysis and reverse-transcription quantitative polymerase chain reaction, respectively. In addition, a correlation analysis of ADAM33 expression and E-cadherin/vimentin expression was performed. A wound healing migration assay and a cell invasion assay were also performed. The results of the cellular morphology, migration and invasion studies suggested that TGF-β1 treatment induced typical EMT changes in HBE cells. In addition, treatment with various concentrations of TGF-β1 significantly increased the protein and mRNA expression levels of ADAM33 and vimentin compared with those in untreated cells. TGF-β1 treatment also decreased the protein and mRNA expression levels of E-cadherin in a dose-dependent manner. By contrast, transfection with siADAM33 promoted the protein expression of E-cadherin and decreased the protein expression of vimentin. Furthermore, ADAM33 and E-cadherin expression levels exhibited a significant negative correlation, whereas ADAM33 and vimentin were positively correlated. In conclusion, the results suggested that TGF-β1 enhances ADAM33 expression in airway epithelial cells, and that ADAM33 induces the EMT of airway epithelial cells, thus participating in airway remodeling in asthma.

The effect and underlying mechanism of miRNA-506 regulating TGF-β1-induced endothelial-mesenchymal transition of human aortic endothelial cell lysates

Objective To investigate the effect and the underlying mechanism of miRNA(miR)-506 regulating transforming growth factor-β1 (TGF-β1)-induced endothelial-mesenchymal transition of human aortic endothelial cell lysates. Methods In this study, miR-506 and inhibitory members of the ASPP family (iASPP) were selected as the study objects. First, the expression of miR-506 in human aortic endothelial cell lysates (HAEC) lysate was detected under different concentrations of TGF-β1. The expression of platelet endothelial cell adhesion molecule-1 (CD31), endothelium-cadherin (VE-cadherin), fibroblast-specific protein-1 (FSP1), α-smooth muscle actin (α-SMA) and N-cadherin was determined after miR-506 mimics/mimic negative control (NC) transfection into HAEC treated with or without TGF-β1. Then the expression levels of iASPP in miRNA-506 mimics/mimic NC-transfected HAECs treated with or without TGF-β1 were determined. Finally, the protein expression of CD31, VE-cadherin, FSP1 and α-SMA in HAEC transfected with miR-506 mimics/mimic NC was determined after iASPP overexpression. Results TGF-β1 can induce human aortic endothelial cell mesenchymal transition, down-regulate CD31 and VE-cadherin expression while up-regulate FSP1, α-SMA and N-cadherin expression; TGF-β1 inhibited miR-506 expression, promoted iASPP expression; miR-506 transfection into HAECs up-regulated CD31 and VE-cadherin e-xpression while up-regulate FSP1, α-SMA and N-cadherin expression; miR-506 could downregulate the expression of iASPP; the overexpression of iASPP increased the expression of FSP1, α-SMA and N-cadherin, down-regulated CD31 and VE-cadherin and promoted the transformation of human aortic endothelial cells. Conclusions The transfection of iASPP plasmid promoted the endothelial-mesenchymal transition of human aortic endothelial cell lysates. The transfection of miR-506 inhibited the expression of iASPP and inhibited the endothelial-mesenchymal transition of human aortic endothelial cell lysates induced by TGF-β1. Key words: MicroRNAs; Intracellular signaling peptides and proteins; Tumor suppressor protein p53; Transforming growth factor beta1; Aorta; Endothelial cells; Biotransformation

Effects of TGF-β1 on plasminogen activation in human dental pulp cells: Role of ALK5/Smad2, TAK1 and MEK/ERK signalling.

Transforming growth factor-β1 (TGF-β1) plays an important role in the pulpal repair and dentinogenesis. Plasminogen activation (PA) system regulates extracellular matrix turnover. In this study, we investigated the effects of TGF-β1 on PA system of dental pulp cells and its signalling pathways. Dental pulp cells were treated with different concentrations of TGF-β1. MTT assay, reverse transcription-polymerase chain reaction, Western blotting and enzyme-linked immunosorbant assay (ELISA) were used to detect the effect of TGF-β1 on cell viability, mRNA and protein expression of urokinase-type plasminogen activator (uPA), uPA receptor (uPAR), plasminogen activator inhibitor-1 (PAI-1) as well as their secretion. The phosphorylation of Smad2 and TAK1 was analysed by Pathscan ELISA or Western blotting. Cells were pretreated with SB431542 (ALK5/Smad2/3 inhibitor), 5z-7-oxozeaenol (TAK1 inhibitor) and U0126 (MEK/ERK inhibitor) for examining the related signalling. TGF-β1 slightly inhibited cell growth that was reversed by SB431542. TGF-β1 upregulated both RNA and protein expression of PAI-1 and uPAR, whereas it downregulated uPA expression. Accordingly, TGF-β1 stimulated PAI-1 and soluble uPAR (suPAR) secretion of pulp cells, whereas uPA secretion was inhibited. TGF-β1 induced the phosphorylation of Smad2 and TAK1. In addition, SB431542, 5z-7-oxozeaenol and U0126 attenuated the TGF-β1-induced secretion of PAI-1 and suPAR. These results indicate that TGF-β1 is possibly involved in the repair/regeneration and inflammatory processes of dental pulp via regulation of PAI-1, uPA and uPAR. These effects of TGF-β1 are related to activation of ALK5/Smad2, TAK1 and MEK/ERK signalling pathways. Clarifying the signal transduction for the effects of TGF-β1 is helpful for pulpo-dentin regeneration and tissue engineering. Copyright © 2016 John Wiley & Sons, Ltd.

The mechanism of epithelial-mesenchymal transition induced by TGF-β1 in neuroblastoma cells.

Neuroblastoma is the second most common extracranial malignant solid tumor that occurs in childhood, and metastasis is one of the major causes of death in neuroblastoma patients. The epithelial-mesenchymal transition (EMT) is an important mechanism for both the initiation of tumor invasion and subsequent metastasis. Therefore, this study investigated the mechanism by which transforming growth factor (TGF)-β1 induces EMT in human neuroblastoma cells. Using quantitative RT-qPCR and western blot analyses, we found that the mRNA and protein expression levels of E-cadherin were significantly decreased, whereas that of α-SMA was significantly increased after neuroblastoma cells were treated with different concentrations of TGF-β1. A scratch test and Transwell migration assay revealed that cell migration significantly and directly correlated with the concentration of TGF-β1 indicating that TGF-β1 induced EMT in neuroblastoma cells and led to their migration. Inhibiting Smad2/3 expression did not affect the expression of the key molecules involved in EMT. Further investigation found that the expression of the glioblastoma transcription factor (Gli) significantly increased in TGF-β1-stimulated neuroblastoma cells undergoing EMT, accordingly, interfering with Gli1/2 expression inhibited TGF-β1-induced EMT in neuroblastoma cells. GANT61, which is a targeted inhibitor of Gli1 and Gli2, decreased cell viability and promoted cell apoptosis. Thus, TGF-β1 induced EMT in neuroblastoma cells to increase their migration. Specifically, EMT induced by TGF-β1 in neuroblastoma cells did not depend on the Smad signaling pathway, and the transcription factor Gli participated in TGF-β1-induced EMT independent of Smad signaling.

TGF-β1 regulating miR-205/miR-195 expression affects the TGF-β signal pathway by respectively targeting SMAD2/SMAD7.

Transforming growth factor-β (TGF-β) proteins are important cytokines in the occurrence and development of tumors. However, its neural functions in glioma are still not understood. In the present study, we evaluated the effects of TGF-β1 on glioma cell line U87. miR-205 and miR-195 were involved in TGF-β1 signaling pathway. Quantitative real-time PCR was used to detect miR-205 and miR-195 levels in human glioma tissue samples and U87 cells treated with different concentrations of TGF-β1. Enzyme-linked immunosorbent assay (ELISA) was performed to determine TGF-β1 in the glioma patients peripheral blood. In vitro, U87 cells were transfected with mimics or inhibitors of miR-205 and miR-195. SMAD proteins were assayed by western blotting. Luciferase assay and co-immunoprecipitation (Co-IP) were used to determine the relationships between miR-205 and SMAD2, miR-195 and SMAD7. Effects of miR-205 and miR-195 on glioma cell proliferation and invasion using colony forming and cell migration assays. It was shown that miR-205 was decreased in glioma tissue, but miR-195 and TGF-β1 was increased. In addition, TGF-β1 concentration was negatively correlated with miR-205 mRNA level, but positively correlated with miR-195 mRNA. In addition, miR-205 was downregulated and miR-195 was upregulated by TGF-β1 in a dose-dependent manner. miR-205 and miR-195 targeted and inhibited SMAD2 and SMAD7 expression, respectively, in U87. High expression of miR-205 but not miR-195 reduced SMAD2 and SMAD4 heteromer formation. In addition, it was also shown that miR-205 overexpression inhibited U87 proliferation and invasion efficiently. All the results suggested that miR-205 and miR-195 participated in the TGF-β1 signaling pathway and showed opposite effects in glioma. These findings contribute to the understanding of TGF-β1 function in glioma.

Phenotype transformation of immortalized NCM460 colon epithelial cell line by TGF-β1 is associated with chromosome instability.

Transforming growth factor-β1 (TGF-β1) within tumor microenvironment has a pivotal function in cancer initiation and tumorigenesis, and hence this study was to observe the malignant transformation induced by TGF-β1 in an immortalized colon epithelial cell line NCM460 for better understanding the mechanisms of colon carcinogenesis. Immortalized colon epithelial cell line NCM460 was used as the model of this study, and was treated with different concentrations of TGF-β1 for different time. Then, immunofluorescence was performed to observe the change of phenotype hallmarks including adherent junction protein E-cadherin, cytoskeleton protein vimentin, and tight junction marker ZO-1, western blotting analysis was performed to detect the expression of the above three markers and two transcription factors (Snail and Slug) involved in the transformation by TGF-β1. In addition, chromosome instability (CHI) including analysis of DNA-ploid was detected by flow cytometry. Our results revealed significant loss or reduction of ZO-1 and E-cadherin, and robust emergence of vimentin in the cell line NCM460 after a 15-, 20-, and 25-day treatment with 10ng/ml TGF-β1. Interestingly, 20 and 25days after stimulation with 5ng/ml TGF-β1, expression of E-cadherin and ZO-1 revealed a pattern roughly similar to that of 10ng/ml TGF-β1, especially, both expressions was vanished and vimentin expression was dramatically increased at days 25 after TGF-β1 stimulation. After a stimulation with 10ng/ml TGF-β1 for 15, 20, and 25days, the levels of Snail and Slug expression in the cells were significantly up-regulated, compared with the cells treated with TGF-β1 inhibitor LY364947, PBS or balnk control (P<0.01). Our results found that many abnormal mitotic patterns including lagging chromosomes and anaphase bridges in NCM460 cells were induced by TGF-β1 after its stimulation for 15, 20, and 25days. Very few mitotic cells with treatment of PBS for 15, 20 and 25days were non-diploid whose DNA content was greater or less than 4N, but these cells were significantly increased after exposure to TGF-β1 for 15, 20, and 25days, which was associated with the induction of hypo-diploid, hyper-diploid, and poly-diploid (P<0.05).These data indicate that TGF-β1 induces a phenotypic transformation of normal colon epithelium similar to its pro-tumoral behaviors in TME, involving in alteration of chromosome stability.

Effects of TGF-β1 on the Proliferation and Apoptosis of Human Cervical Cancer Hela Cells In Vitro.

To investigate the effects of TGF-β1 on the proliferation and apoptosis of cervical cancer Hela cells in vitro. Human cervical cancer Hela cells were cultured in vitro and divided into the experimental and control groups. In the experimental groups, Hela cells were stimulated with different concentrations of TGF-β1 (0.01, 0.1, 1, and 10ng/mL), while Hela cells cultured in serum-free medium without TGF-β1 were used as controls. The CCK8 method was adopted to detect the effect of TGF-β1 on Hela cell proliferation, and flow cytometry was used to determine cell apoptosis 72h after TGF-β1 treatment. Compared with the control group, the CCK-8 tests showed that different concentrations of TGF-β1 had no obvious effect on Hela cell proliferation 24h after treatment (P>0.05). However, upon 48 or 72h of treatment, TGF-β1 significantly inhibited the proliferation of Hela cells in a time- and dose-dependent manner (P<0.05). The flow cytometry results indicated that TGF-β1 influenced the apoptosis of human cervical cancer Hela cells in a dose-dependent manner after 72h of treatment (P<0.05). TGF-β1 significantly inhibited the growth and induced the apoptosis of human cervical Hela cells in vitro.

The expression of periostin in dental pulp cells

Background and objectiveDental pulp repair is a common process triggered by microbial and mechanical challenges. Matricellular modulators, such as periostin, are key for extracellular matrix stability and tissue healing. In the scope of the dental pulp, periostin expression has been reported during development and active dentinogenesis. However, the specific dental pulp cell population capable of expressing periostin in response to known regulators has not been clearly defined. Among the different relevant cell populations (i.e., stem cells, fibroblasts and pre-odontoblasts) potentially responsible for periostin expression in the dental pulp, this study aimed to determine which is the primary responder to periostin regulators. MethodsHuman dental pulp stem cells (DPSCs), human dental pulp fibroblasts (DPFs), and rat odontoblast-like cells (MDPC-23) were treated with different concentrations of TGF-β1 or different regimens of biomechanical stimulation to evaluate periostin expression by qRT-PCR, Western blot and ELISA. Statistical analyses were performed by Student's t-test and ANOVA with Fisher's LSD post hoc tests (p≤0.05). ResultsDPSC and MDPC-23 showed a statistically significant increase in periostin mRNA expression after exposure to TGF-β1 for 48h. TGF-β1 also up-regulated periostin protein levels in DPSC. However, periostin significantly down-regulated protein expression in DPF. Different regimens of biomechanical stimulation showed different patterns in protein and mRNA periostin expression. ConclusionsExpression of periostin was identified in each of the analysed dental pulp cell lines, which can be regulated by TGF-β1 and biomechanical stimulation. Overall, DPSCs are the most responsive cells to stimulation.

Transforming growth factor beta 1 up-regulates CD169 (sialoadhesin) expression on monocyte-derived dendritic cells: role in HIV sexual transmission.

Recent data describe CD169 (also called sialoadhesin or Siglec-1) as the main HIV-1 receptor expressed by mucosal dendritic cells involved in the capture of the virus and its transmission to target cells. In this study, we investigated the effect of transforming growth factor beta 1 (TGF-β1), a cytokine found in abundance in semen, on the expression of CD169 on dendritic cells in order to characterize its potential role in the capture of HIV-1 particles by these antigen-presenting cells. Monocyte-derived dendritic cells (MDDCs) were cultured in the presence of lipopolysaccharide, pro-inflammatory cytokines [interleukin (IL)-1β and tumor necrosis factor alpha (TNF-α)] or different concentrations of TGF-β1, and analyzed for maturation marker and Siglec expression. The ability of MDDCs to capture HIV particles following the different treatments was also analyzed. TGF-β1 treatment promotes a significant increase of CD169 expression on MDDCs. This effect was specific since neither DC-SIGN nor other Siglec expressions were changed. The CD169 increase was due to a de-novo synthesis as evidenced by Western blot experiment. This up-regulation was well correlated to the concentration of TGF-β1 and associated with an increase of the MDDC ability to bind HIV particles. Interestingly, this phenomenon was independent of the maturation status of MDDCs. This study demonstrates that the most abundant cytokine present in semen (TGF-β1) is able to enhance specifically the expression of an important molecule (CD169) involved in the capture and transmission of HIV-1 particles from the mucosal lumen to the submucosal compartment. Our results suggest that this mechanism may play a relevant role in sexual HIV transmission.

Transforming growth factor-β1 deteriorates microrheological characteristics and motility of mature dendritic cells in concentration-dependent fashion

Dendritic cells (DCs) are potent and specialized antigen-presenting cells that play a crucial role in initiating and amplifying both the innate and adaptive immune responses. Tumor cells can escape from immune attack by secreting suppressive cytokines which solely or cooperatively impair the immune function and microrheological properties of DCs. However, the underlying mechanisms are not fully defined. Transforming growth factor-β1 (TGF-β1) has been identified as a major cytokine in the tumor microenvironment. To determine the effects of TGF-β1 on mature DCs (mDCs) from microrheological viewpoint, cells were treated with different concentrations of TGF-β1. The results showed that the impaired microrheological parameters, including osmotic fragility, electrophoretic mobility, deformability, membrane fluidity, F-actin organization and so on, as well as motilities of mDCs relied heavily on TGF-β1 concentration. Moreover, these changes were correlated with the expression levels of fascin1, cofilin1, phosphorylated cofilin1 and profilin, this could be one of the crucial aspects of immune escape mechanisms of tumors, hinting that the signal pathway of TGF-β1 should be blocked in appropriate way before performing DCs-based immunotherapy against cancer. It is clinically important to understand the biological behavior of DCs and immune escape mechanism of tumor as well as how to improve efficiency of the anti-tumor therapy based on DCs.