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

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

ITE promotes hypoxia-induced transdifferentiation of human pulmonary arterial endothelial cells possibly by activating transforming growth factor-β/Smads and MAPK/ERK pathways

This study aimed to investigate the transdifferentiation of human pulmonary arterial endothelial cells (HPAECs) into smooth muscle like (SM-like) cells under hypoxic conditions and reveal the role of endogenous small molecular compound 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylicacid methyl ester (ITE) in this process. HPAECs were treated by hypoxia and hypoxia + ITE with different durations. The endothelial markers (CD31 and VE-cad) and smooth muscle markers (α-SMA, SM22α, and OPN) were investigated by immunofluorescence double staining, and their expressions, along with the differentiation regulators transforming growth factor-β (TGF-β) ligands and downstream signals including TGF-β1, bone morphogenetic protein (BMP2), BMP9, Samd2/3, ERK, and p38 MAPK, were determined by Western blot analysis. The viability and proliferation of HPAECs were detected by Cell Counting Kit-8 (CCK-8) method and bromodeoxyuridine (BrdU) assays. As a result, hypoxia induced HPAECs transdifferentiation from paving-stone-like into polygonal or spindle cells, whose number increased greatly after additional ITE stimulation for 7 days. Compared with the normoxic HPAECs, the expression of endothelial markers reduced and smooth muscle markers were enhanced with the extension of hypoxia + ITE treatment, and meanwhile the cell viability increased significantly. Hypoxia could promote expression of TGF-β1 protein rather than BMP2 and BMP9, and regulate phosphorylation levels of Samd2/3, ERK and p38 MAPK in different manners. In conclusion, ITE can promote the hypoxia-induced transdifferentiation of HPAECs into SM-like cells via TGF-β/Smads and MAPK/ERK pathways.     

Activation of extracellular signal-regulated kinase by TGF-β1 via TβRII and Smad7 dependent mechanisms in human bronchial epithelial BEP2D cells

Transforming growth factor-β1 (TGF-β1) can activate mitogen-activated protein kinases (MAPKs) in many types of cells. The mechanism of this activation is not well elucidated. Here, we explore the role of TGF-β/Smads signaling compounds in TGF-β1-mediated activation of extracellular signal-regulated kinase (ERK) MAPK in human papillomavirus (HPV)-18 immortalized human bronchial epithelial cell line BEP2D and the role of TGF-β1-induced phosphorylation of ERK in proliferation and apoptosis of BEP2D. The cell models of siRNA-mediated silencing of TGF-β receptor type II (TβRII), Smad2, Smad3, Smad4, and Smad7 were employed in this study. Our results demonstrate that TGF-β1 activates ERK in a time-dependent manner with a maximum effect at 60 min; overexpression of Smad7 increased this TGF-β1-mediated phosphorylation of the ERK; and siRNA-mediated silencing of TβRII, Smad3, Smad4, and Smad7 abrogated this effect. Moreover, we observed that overexpression of Smad7 restored TGF-β1-mediated ERK phosphorylation in Smad4 knockdown cells but not in TβRII knockdown cells. In BEP2D cells, TGF-β1 treatment effectively inhibited cells’ proliferation and induced their apoptosis. Pretreatment with U0126, an inhibitor of ERK1/2, significantly enhanced the TGF-β1-mediated antiproliferative and apoptosis induction effects in BEP2D cells. These data revealed that TβRII and Smad7 play the critical roles in TGF-β1-mediated activation of ERK; Smad3 and Smad4 can play an indirect role through up-regulating Smad7 expression; and TGF-β1-induced phosphorylation of ERK may participate in BEP2D cell proliferation and apoptosis regulation.     

Micro-computed tomography of pulmonary fibrosis in mice induced by adenoviral gene transfer of biologically active transforming growth factor-β1

Background

Micro-computed tomography (micro-CT) is a novel tool for monitoring acute and chronic disease states in small laboratory animals. Its value for assessing progressive lung fibrosis in mice has not been reported so far. Here we examined the importance of in vivo micro-CT as non-invasive tool to assess progression of pulmonary fibrosis in mice over time.

Methods

Pulmonary fibrosis was induced in mice by intratracheal delivery of an adenoviral gene vector encoding biologically active TGF-ß1 (AdTGF-ß1). Respiratory gated and ungated micro-CT scans were performed at 1, 2, 3, and 4 weeks post pulmonary adenoviral gene or control vector delivery, and were then correlated with respective histopathology-based Ashcroft scoring of pulmonary fibrosis in mice. Visual assessment of image quality and consolidation was performed by 3 observers and a semi-automated quantification algorithm was applied to quantify aerated pulmonary volume as an inverse surrogate marker for pulmonary fibrosis.

Results

We found a significant correlation between classical Ashcroft scoring and micro-CT assessment using both visual assessment and the semi-automated quantification algorithm. Pulmonary fibrosis could be clearly detected in micro-CT, image quality values were higher for respiratory gated exams, although differences were not significant. For assessment of fibrosis no significant difference between respiratory gated and ungated exams was observed.

Conclusions

Together, we show that micro-CT is a powerful tool to assess pulmonary fibrosis in mice, using both visual assessment and semi-automated quantification algorithms. These data may be important in view of pre-clinical pharmacologic interventions for the treatment of lung fibrosis in small laboratory animals.     

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

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

Activation of PPAR-α induces cell cycle arrest and inhibits transforming growth factor-β1 induction of smooth muscle cell phenotype in 10T1/2 mesenchymal cells

Transforming growth factor-β1 (TGF-β1) regulates the cell cycle and the differentiation of mesenchymal cells into smooth muscle cells (SMCs). However, the precise intracellular signaling pathways involved in these processes have not been fully clarified. It has also been shown that there is an increase in TGF-β1 expression in human atherosclerotic plaques. Furthermore, peroxisome proliferator-activated receptors (PPARs) and their agonists have recently gained more attention in the study of the pathogenesis of atherosclerosis. In this study, we examined the role of PPARs in the TGF-β1-mediated cell cycle control and SMC phenotypic modulation of C3H10T1/2 (10T1/2) mesenchymal cells. The results showed the following: (1) the PI3K/Akt/p70S6K signaling cascade is involved in TGF-β1-induced differentiation of 10T1/2 cells into cells with a SMC phenotype. (2) PPAR-α agonists (i.e., WY14,643 and clofibrate), but not a PPAR-δ/β agonist (GW501516) or PPAR-γ agonist (troglitazone), inhibit TGF-β1-induced SMC markers and the DNA binding activity of serum response factor (SRF) in 10T1/2 cells. (3) WY14,643 and clofibrate inhibit the TGF-β1 activation of the Smad3/Akt/P70S6K signaling cascade. (4) TGF-β1-induced cell cycle arrest at the G₀/G₁ phases is mediated by Smad3 in 10T1/2 cells. (5) The PPAR-α-mediated 10T1/2 cell cycle arrest at the G₀/G₁ phases is TGF-β receptor independent. These results suggest that PPAR-α mediates cell cycle control and TGF-β1-induced SMC phenotypic changes in 10T1/2 cells.     

Alteration of protein glycosylation in human hepatic stellate cells activated with transforming growth factor-β1

Although aberrant glycosylation of human glycoproteins is related to liver fibrosis that results from chronic damage to the liver in conjunction with the activation of hepatic stellate cells (HSCs), little is known about the precision alteration of protein glycosylation referred to the activation of HSCs by transforming growth factor-β1 (TGF-β1). The human HSCs, LX-2 were activated by TGF-β1. The lectin microarrays were used to probe the alteration of protein glycosylation in the activated HSCs compared with the quiescent HSCs. Lectin histochemistry was used to further validate the lectin binding profiles and assess the distribution of glycosidic residues in cells. As a result, 14 lectins (e. g. AAL, PHA-E, ECA and ConA) showed increased signal while 7 lectins (e. g. UEA-I and GNA) showed decreased signal in the activated LX-2 compared with the quiescent LX-2. Meanwhile, AAL, PHA-E and ECA staining showed moderate binding to the cytoplasma membrane in the quiescent LX-2, and the binding intensified in the same regions of the activated LX-2. In conclusion, the precision alteration of protein glycosylation related to the activation of the HSCs may provide useful information to find new molecular mechanism of HSC activation and antifibrotic therapeutic strategies.     

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.     

Activation of peroxisome proliferation–activated receptor-γ inhibits transforming growth factor-β1-induced airway smooth muscle cell proliferation by suppressing Smad–miR-21 signaling

The aims of the current study were to examine the signaling mechanisms for transforming growth factor-β1 (TGF-β1)-induced rat airway smooth muscle cell (ASMC) proliferation and to determine the effect of activation of peroxisome proliferation–activated receptor-γ (PPAR-γ) on TGF-β1-induced rat ASMC proliferation and its underlying mechanisms. TGF-β1 upregulated microRNA 21 (miR-21) expression by activating Smad2/3, and this in turn downregulated forkhead box O1 (FOXO1) mRNA expression. In addition, TGF-β1–Smad–miR-21 signaling also downregulated phosphatase and tensin homolog deleted on chromosome ten (PTEN) expression and thus de-repressed the PI3K–Akt pathway. Depletion of PTEN reduced the nuclear FOXO1 protein level without affecting its mRNA level. Inhibition of the PI3K–Akt pathway or proteasome function reversed PTEN knockdown-induced nuclear FOXO1 protein reduction. Our study further showed that loss of FOXO1 increased cyclin D1 expression, leading to rat ASMC proliferation. Preincubation of rat ASMCs with pioglitazone, a PPAR-γ activator, blocked TGF-β1-induced activation of Smad2/3 and its downstream targets changes of miR-21, PTEN, Akt, FOXO1, and cyclin D1, resulting in the inhibition of rat ASMC proliferation. Our study suggests that the activation of PPAR-γ inhibits rat ASMC proliferation by suppressing Smad–miR-21 signaling and therefore has a potential value in the prevention and treatment of asthma by negatively modulating airway remodeling.     

ROS directly activates transforming growth factor β type 1 receptor signalling in human vascular smooth muscle cells

BackgroundWidely used NAPDH oxidase (Nox) inhibitor, apocynin is a prodrug that needs to be converted to its pharmacologically active form by myeloperoxidase. In myeloperoxidase deficient non phagocytic cells such as vascular smooth muscle cells (VSMCs) apocynin stimulates the production of ROS. ROS is generated by the activation of many signalling pathways, thus we have used apocynin as a pharmacological tool to characterise the role of endogenous ROS in activating the transforming growth factor beta receptor (TGFBR1) without the activation of other pathways.MethodsThe in vitro study utilized human VSMCs. Western blotting and quantitative real time PCR were performed to assess signalling pathways and gene expression, respectively. Intracellular ROS levels was measured using fluorescence detection assay.ResultsTreatment with apocynin of human VSMCs stimulated ROS production and the phosphorylation of TGFBR1 and subsequent activation of TGFBR1 signalling leading to the formation of phosphorylated Smad2 which consequently upregulates the mRNA expression of glycosaminoglycan synthesizing enzyme.ConclusionsThese findings outline a specific involvement of ROS production in TGFBR1 activation. Furthermore, because apocynin stimulates Nox and ROS production, apocynin must be used with considerable care in vitro as its actions clearly extend beyond the stimulation of Nox enzymes and it has consequences for cellular signalling.General significanceApocynin can stimulate Nox leading to the production of ROS and the outcome is completely dependent upon the redox properties of the cell.     

Inhibition of insulin-like growth factor-1 (IGF-1) expression by prolonged transforming growth factor-β1 (TGF-β1) administration suppresses osteoblast differentiation

TGF-β1 can regulate osteoblast differentiation not only positively but also negatively. However, the mechanisms of negative regulation are not well understood. We previously established the reproducible model for studying the suppression of osteoblast differentiation by repeated or high dose treatment with TGF-β1, although single low dose TGF-β1 strongly induced osteoblast differentiation. The mRNA expression and protein level of insulin-like growth factor-1 (IGF-1) were remarkably decreased by repeated TGF-β1 administration in human periodontal ligament cells, human mesenchymal stem cells, and murine preosteoblast MC3T3-E1 cells. Repeated TGF-β1 administration subsequently decreased alkaline phosphatase (ALP) activity and mRNA expression of osteoblast differentiation marker genes, such as RUNX2, ALP, and bone sialoprotein (BSP). Additionally, repeated administration significantly reduced the downstream signaling pathway of IGF-1, such as Akt phosphorylation in these cells. Surprisingly, exogenous and overexpressed IGF-1 recovered ALP activity and mRNA expression of osteoblast differentiation marker genes even with repeated TGF-β1 administration. These facts indicate that the key mechanism of inhibition of osteoblast differentiation induced by repeated TGF-β1 treatment is simply due to the down-regulation of IGF-1 expression. Inhibition of IGF-1 signaling using small interfering RNA (siRNA) against insulin receptor substrate-1 (IRS-1) suppressed mRNA expression of RUNX2, ALP, BSP, and IGF-1 even with single TGF-β1 administration. This study showed that persistence of TGF-β1 inhibited osteoblast differentiation via suppression of IGF-1 expression and subsequent down-regulation of the PI3K/Akt pathway. We think this fact could open the way to use IGF-1 as a treatment tool for bone regeneration in prolonged inflammatory disease.     

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.     

Role of protein kinase C δ in ER stress and apoptosis induced by oxidized LDL in human vascular smooth muscle cells

During atherogenesis, excess amounts of low-density lipoproteins (LDL) accumulate in the subendothelial space where they undergo oxidative modifications. Oxidized LDL (oxLDL) alter the fragile balance between survival and death of vascular smooth muscle cells (VSMC) thereby leading to plaque instability and finally to atherothrombotic events. As protein kinase C δ (PKCδ) is pro-apoptotic in many cell types, we investigated its potential role in the regulation of VSMC apoptosis induced by oxLDL. We found that human VSMC silenced for PKCδ exhibited a protection towards oxLDL-induced apoptosis. OxLDL triggered the activation of PKCδ as shown by its phosphorylation and nuclear translocation. PKCδ activation was dependent on the reactive oxygen species generated by oxLDL. Moreover, we demonstrated that PKCδ participates in oxLDL-induced endoplasmic reticulum (ER) stress-dependent apoptotic signaling mainly through the IRE1α/JNK pathway. Finally, the role of PKCδ in the development of atherosclerosis was supported by immunohistological analyses showing the colocalization of activated PKCδ with ER stress and lipid peroxidation markers in human atherosclerotic lesions. These findings highlight a role for PKCδ as a key regulator of oxLDL-induced ER stress-mediated apoptosis in VSMC, which may contribute to atherosclerotic plaque instability and rupture.     

Control of nitric oxide synthase expression by transforming growth factor-β: Implications for homeostasis

Production of nitric oxide (NO) can be stimulated by inflammatory cytokines and bacterial lipopolysaccharide (LPS) in mammalian cells via an inducible nitric oxide synthase (iNOS). Conversely, the transforming growth factor-βs (TGF-βs) suppress NO production by reducing iNOS expression. Production of NO leads to disparate consequences, some beneficial and some damaging to the host, depending on the cell and context in which iNOS is induced. The TGF-βs counter these NO-mediated processes in macrophages, cardiac myocytes, smooth muscle cells, bone marrow cells, and retinal pigment epithelial cells. Autocrine or paracrine production of TGF-β may thus serve as a physiological counterbalance for iNOS expression, a mechanism which may be subverted by pathogens and tumors for their own survival. A greater understanding of the mechanisms and consequences of NO and TGF-β production may lead to effective therapeutic strategies in various diseases.     

Urotensin upregulates transforming growth factor-β1 expression of asthma airway through ERK-dependent pathway

Airway smooth muscle cells (ASMCs) play a key role in the process of asthma airway remodeling. Urotensin II (UII) and transforming growth factor (TGF)-β are potent mitogens for ASMCs proliferation. The study was aimed to determine whether UII-upregulated TGF-β-mediated ASMCs proliferation and extracellular signal-regulated kinase (ERK) was required for such an effect. OVA-sensitized rats were challenged to induce asthma. Lung morphology and airway dynamic parameters were monitored. ASMCs from control and asthma rats were purified for the measurement of UII and TGF-β1 expression. In vitro experiments were conducted to determine the direct effect of UII on TGF-β1 expression by ASMCs. Finally, U0126, an ERK inhibitor was used to examine the role of ERK pathway in UII mediated TGF-β1 upregulation. We found that both UII and TGF-β1 were upregulated in asthma lung tissues. In vitro study on ASMCs further revealed that UII may render its effect on ASMCs cells through the upregulation of TGF-β1. Data also supported the conclusion that ERK pathway was required, but not sufficient in UII-induced TGF-β1 upregulation. The current study provides new evidence that UII is involved in the TGF-β mediated mitogenic effect on ASMCs. UII, at least partially, uses ERK pathway to render such effect.     

Inhibition of transforming growth factor-β2 expression with phosphorothioate antisense oligonucleotides in U937 cells

Four types of phosphorothioate antisense oligonucleotides for transforming growth factor-β2 were synthesized and tested for their antisense activity in U937 cells. The full-length phosphorothioate modified antisense analogues exhibited the highest inhibitory effects on the transforming growth factor-β2 expression in U937 cells.