RNA-Seq reveals placental growth factor regulates the human retinal endothelial cell barrier integrity by transforming growth factor (TGF-β) signaling

Molecular and Cellular Biochemistry - Previous studies have indicated that long non-coding RNAs (lncRNAs) were closely related to diabetes. In this study, we aimed to explore the possible role and...     

Activation of phospholipase D activity in transforming growth factor—beta—induced cell growth inhibition

Cells regulate phospholipase D(PLD) activity in response to numerous extracellular signals.Here,we investigated the involvement of PLD activity in transforming growth factor-β(TGF-β1)-mediated growth inhibition of epithelial cells.TGF-β1)-mediated growth inhibition of epithelial cells.TGF-β1 inhibits the growth of MDCK,Mv1Lu,and A-549 cells.In the presence of 0.4% butanol,TGF-β1 induces an increase in the formation of phosphatidylbutanol,a unique product catalyzed by PLD.TGF-β1 also induces an increase in phosphatidic acid (PA) level in A-549 and MDCK cells.TGF-β1 induces an increase in the levels of DAG labeled with [^3H]-myristic acid in A-549 and MDCK cells but not in Mv1Lu cells.No increase of DAG was observed in cells prelabeled with [^3H]-arachidonic acid.The data presented suggest that PLD activation is involved in the TGF-β1-induced cell growth inhibition.     

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.     

Effect of transforming growth factor beta (TGF-β) on ATP citrate lyase in isolated hepatocytes

Summary Transforming growth factor beta (TGF-) activates ATP citrate lyase in freshly isolated rat liver hepatocytes in a time dependent manner. Maximal stimulation of the enzyme occurred with less than thirty minutes of incubation of the cells with TGF-. The half maximal effect on the enzyme determined in hepatocytes incubated with TGF- for 10 min at 37°C was elicited by TGF- concentrations in the 10^–11 – 10^–12 M range. The potential role of TGF- stimulation of ATP citrate lyase activity in new membrane synthesis is discussed.     

In vitro secretion of transforming growth factor alpha (TGF-α): A comparison of the A431 cell line with three human oesophageal squamous cell carcinoma lines

Transforming growth factor alpha (TGF-) is a single chain polypeptide which exists in a variety of forms differing in molecular weight. These forms are variously present in normal and neoplastic cells. Of particular interest are TGF-'s well-known mitogenic properties. The transition from a normal to a neoplastic cellular state results from signalling defects that may depend upon,iter alia, abonormal levels of expression and secretion of TGF-. It is known that the secretion of TGF- may be enhanced appreciably by agents such as phorbol 12-myristate 13-acetate (PMA), serum factors and epidermal growth factor (EGF). Here, we compare the efficacy of these three agents in the elevation of TGF- secretion in the well studied A431 cell line with their previously undocumented efficacy in certain interesting, but little known, human oesophageal squamous cell carcinoma (SCC) lines.     

Heterobicyclic inhibitors of transforming growth factor beta receptor I (TGFβRI)

The TGFβ-TGFβR signaling pathway has been reported to play a protective role in the later stages of tumorigenesis via increasing immunosuppressive Treg cells and facilitating the epithelial to mesenchymal transition (EMT). Therefore, inhibition of TGFβR has the potential to enhance antitumor immunity. Herein we disclose the identification and optimization of novel heterobicyclic inhibitors of TGFβRI that demonstrate potent inhibition of SMAD phosphorylation. Application of structure-based drug design to the novel pyrrolotriazine chemotype resulted in improved binding affinity (Ki apparent?=?0.14?nM), long residence time (T_1/2?>?120?min) and significantly improved potency in the PSMAD cellular assay (IC₅₀?=?24?nM). Several analogs inhibited phosphorylation of SMAD both in vitro and in vivo. Additionally, inhibition of TGFβ-stimulated phospho-SMAD was observed in primary human T cells.     

Roles of N-acetylglucosaminyltransferase III in epithelial-to-mesenchymal transition induced by transforming growth factor β1 (TGF-β1) in epithelial cell lines

The epithelial-to-mesenchymal transition (EMT) plays crucial roles in embryonic development, wound healing, tissue repair, and cancer progression. Results of this study show how transforming growth factor β1 (TGF-β1) down-regulates expression of N-acetylglucosaminyltransferase III (GnT-III) during EMT-like changes. Treatment with TGF-β1 resulted in a decrease in E-cadherin expression and GnT-III expression, as well as its product, the bisected N-glycans, which was confirmed by erythro-agglutinating phytohemagglutinin lectin blot and HPLC analysis in human MCF-10A and mouse GE11 cells. In contrast with GnT-III, the expression of N-acetylglucosaminyltransferase V was slightly enhanced by TGF-β1 treatment. Changes in the N-glycan patterns on α3β1 integrin, one of the target proteins for GnT-III, were also confirmed by lectin blot analysis. To understand the roles of GnT-III expression in EMT-like changes, the MCF-10A cell was stably transfected with GnT-III. It is of particular interest that overexpression of GnT-III influenced EMT-like changes induced by TGF-β1, which was confirmed by cell morphological changes of phase contrast, immunochemical staining patterns of E-cadherin, and actin. In addition, GnT-III modified E-cadherin, which served to prolong E-cadherin turnover on the cell surface examined by biotinylation and pulse-chase experiments. GnT-III expression consistently inhibited β-catenin translocation from cell-cell contact into the cytoplasm and nucleus. Furthermore, the transwell assay showed that GnT-III expression suppressed TGF-β1-induced cell motility. Taken together, these observations are the first to clearly demonstrate that GnT-III affects cell properties, which in turn influence EMT-like changes, and to explain a molecular mechanism for the inhibitory effects of GnT-III on cancer metastasis.     

Constitutive Smad linker phosphorylation in melanoma: a mechanism of resistance to transforming growth factor-β-mediated growth inhibition

Melanoma cells are resistant to transforming growth factor-β (TGFβ)-induced cell-cycle arrest. In this study, we investigated a mechanism of resistance involving a regulatory domain, called linker region, in Smad2 and Smad3, main downstream effectors of TGFβ. Melanoma cells in culture and tumor samples exhibited constitutive Smad2 and Smad3 linker phosphorylation. Treatment of melanoma cells with the MEK1/2 inhibitor, U0126, or the two pan-CDK and GSK3 inhibitors, Flavopiridol and R547, resulted in decreased linker phosphorylation of Smad2 and Smad3. Overexpression of the linker phosphorylation-resistant Smad3 EPSM mutant in melanoma cells resulted in an increase in expression of p15(INK4B) and p21(WAF1) , as compared with cells transfected with wild-type (WT) Smad3. In addition, the cell numbers of EPSM Smad3-expressing melanoma cells were significantly reduced compared with WT Smad3-expressing cells. These results suggest that the linker phosphorylation of Smad3 contributes to the resistance of melanoma cells to TGFβ-mediated growth inhibition.     

Regulation of autophagy by transforming growth factor-β (TGF-β) signaling

Transforming growth factor-β (TGF-β) has broad impacts on an array of diverse cellular functions including cell growth, differentiation, adhesion, migration, and apoptosis. Perturbations of the TGF-β signaling pathways are involved in progression of various tumors. Autophagy is a pivotal response of normal and cancer cells to environmental stresses and is induced by various stimuli. Otherwise, autophagy has an intrinsic function in tumor suppression. Recently, we demonstrated that TGF-β induces autophagy in hepatocellular carcinoma cells and mammary carcinoma cells. Autophagy activation by TGF-β is mediated through the Smad and JNK pathways. We show that siRNA-mediated knockdown of autophagy genes suppresses the growth inhibitory function of TGF-β and that autophagy activation potentiates TGF-β-mediated induction of proapoptotic genes, Bim and Bmf, in hepatoma cells. In this context, the autophagy pathway might contribute to the growth inhibitory effect of TGF-β, in conjunction with other anti-proliferative pathways downstream of TGF-β signaling. The context and manner by which the TGF-β signaling pathway regulates autophagy have implications for a better understanding of pathological and bidirectional roles of TGF-β signaling pathways in tumorigenesis.     

Comparison of transforming growth factor β and a human tumour-derived suppressor factor

Summary Serum-free supernatants from the human melanoma cell line G361 contain a factor that can potently suppress the generation of tumouricidal lymphokine-activated killer (LAK) cells in response to interleukin-2. To characterise the suppressive factor of tumour origin we performed a number of physicochemical and functional comparisons with another immunosuppressive protein, transforming growth factor (TGF). The bioactivity of tumour-derived suppressor factor (TDSF), assayed by suppression of LAK cell generation, was unaffected by a reducing agent but lost when denatured with a chaotropic agent. In contrast, TGF was inactivated by reduction but not denaturation. TDSF lost bioactivity in conditions of pH less than 4, whereas TGF showed no loss of activity. The TDSF moiety has an estimated pI of 4.3 and a molecular mass of 69–87 kDa. This differs from published values of pI 9.5, and 25 kDa molecular mass for TGF. Anti-TGF antiserum reversed the effects of TGF but did not affect the suppression of LAK cell generation caused by TDSF. These findings provide compelling evidence that the TDSF moiety is not TGF, and may be a novel immunoregulatory cytokine.     

Overexpression of transforming growth factor type?III receptor restores TGF-β1 sensitivity in human tongue squamous cell carcinoma cells

The transforming growth factor type III receptor (TβRIII), also known as β-glycan, is a multi-functional sensor that regulates growth, migration and apoptosis in most cancer cells. We hereby investigated the expression of TβRIII in clinical specimens of tongue squamous cell carcinoma (TSCC) and the underlying mechanism that TβRIII inhibits the growth of CAL-27 human oral squamous cells. The TSCC tissues showed a significant decrease in TβRIII protein expression as detected by immunohistochemistry (IHC) and western blot analysis. Transfection of TβRIII-containing plasmid DNA dramatically promoted TGF-β1 (10 ng/ml)-induced decrease in cell viability, apoptosis and cell arrest at the G₀-/G₁-phase. Moreover, transient overexpression of TβRIII enhanced the TGF-β1-induced cyclin-dependent kinase inhibitor 2b (CDKN2b) and p38 protein activity, but did not affect the activities of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase 1/2 (JNK1/2) in CAL-27 cells. These results suggest overexpression of TβRIII receptor restored TGF-β1 sensitivity in CAL-27 cells, which may provide some new insights on exploiting this molecule therapeutically.     

Characterization of a new human diploid cell line—IMR-91

Summary A human diploid fibroblast cell line has been established from the lung tissue of a male fetus. This has been characterized and frozen away in large quantity. A smaller quantity of fibroblastlike cells from skin has also been established, partially characterized, and placed in frozen storage from the same fetus. This project is in support of the National Institute on Aging research in general cell biology. The present lines designated IMR-91 lung and IMR-91 skin complement the previous human diploid fibroblast culture (IMR-90) established from a female fetus. The lack of random inactivation of one of the two X chromosomes in the present male line reduces the genetic heterogeneity inherent in the female line.     

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.     

Exposure to transforming growth factor-β1 after basic fibroblast growth factor promotes the fibroblastic differentiation of human periodontal ligament stem/progenitor cell lines

Basic fibroblast growth factor (bFGF) is a cytokine that promotes the regeneration of the periodontium, the specialized tissues supporting the teeth. bFGF, does not, however, induce the synthesis of smooth muscle actin alpha 2 (ACTA2), type I collagen (COL1), or COL3, which are principal molecules in periodontal ligament (PDL) tissue, a component of the periodontium. We have suggested the feasibility of using transforming growth factor-β1 (TGFβ1) to induce fibroblastic differentiation of PDL stem/progenitor cells (PDLSCs). Here, we investigated the effect of the subsequent application of TGFβ1 after bFGF (bFGF/TGFβ1) on the differentiation of PDLSCs into fibroblastic cells. We first confirmed the expression of bFGF and TGFβ1 in rat PDL tissue and primary human PDL cells. Receptors for both bFGF and TGFβ1 were expressed in the human PDLSC lines 1-11 and 1-17. Exposure to bFGF for 2 days promoted vascular endothelial growth factor gene and protein expression in both cell lines and down-regulated the expression of ACTA2, COL1, and COL3 mRNA in both cell lines and the gene fibrillin 1 (FBN1) in cell line 1-11 alone. Furthermore, bFGF stimulated cell proliferation of these cell lines and significantly increased the number of cells in phase G2/M in the cell lines. Exposure to TGFβ1 for 2 days induced gene expression of ACTA2 and COL1 in both cell lines and FBN1 in cell line 1-11 alone. BFGF/TGFβ1 treatment significantly up-regulated ACTA2, COL1, and FBN1 expression as compared with the group treated with bFGF alone or the untreated control. This method might thus be useful for accelerating the generation and regeneration of functional periodontium.