Activity identification of ribozyme and U1 snRNA chimeric ribozyme against TGFβ1 in cell-free system and in hepatic stellate cells

Transforming growth factorβ1 (TGFβ1) is known to be intimately involved in many cellular processes. To explore the mechanism of TGFβ1 in these processes, the non-chimeric hammerhead ribozyme and U1 snRNA chimeric ribozyme against TGFβ1 were designed to down-regulate TGFβ1 expression. The activity of non-chimeric ribozyme and U1 snRNA chimeric ribozyme against TGFβ1 in vitro and in activated hepatic stellate cells (HSCs) was detected. Cleavage reactions of both ribozymes in vitro demonstrated that non-chimeric ribozyme possessed better cleavage activity in vitro than U1 snRNA chimeric ribozyme. The further study showed U1 snRNA chimeric ribozyme inhibited TGFβ1 expression more efficiently than non-chimeric ribozyme in transfected HSC cells. So it indicates that the U1 snRNA chimeric ribozyme provides an alternative approach for the research on the precise mechanism of TGFβ1 in many cellular processes and a potential therapeutic candidate for TGFβ1-related diseases.     

AKINβ1 is Involved in the Regulation of Nitrogen Metabolism and Sugar Signaling in Arabidopsis

Sucrose non-fermenting-1-related protein kinase 1 (SnRK1) has been located at the heart of the control of metabolism and development in plants. The active SnRK1 form is usually a heterotrimeric complex. Subcellular localization and specific target of the SnRK1 kinase are regulated by specific beta subunits. In Arabidopsis, there are at least seven genes encoding beta subunits, of which the regulatory functions are not yet clear. Here, we tried to study the function of one beta subunit, AKINβ1. It showed that AKINβ1 expression was dramatically induced by ammonia nitrate but not potassium nitrate, and the investigation of AKINβ1 transgenic Arabidopsis and T-DNA insertion lines showed that AKINβ1 negatively regulated the activity of nitrate ruductase and was positively involved in sugar repression in early seedling development. Meanwhile AKINβ1 expression was reduced upon sugar treatment (including mannitol) and did not affect the activity of sucrose phos-phate synthase. The results indicate that AKINβ1 is involved in the regulation of nitrogen metabolism and sugar signaling.     

TGF-β1-promoted epithelial-to-mesenchymal transfor mation and cell adhesion contribute to TGF-β1-enhanced cell migration in SMMC-7721 cells

Transforming growth factor-bl (TGF-β1), a multi-function polypeptide, is a double-edged sword in cancer. For some tumor cells, TGF-β1 is a potent growth inhibitor and apoptosis inducer. More commonly, TGF-β1 losesits growth-inhibitory and apoptosis-inducing effects, but stimulates the metastatic capacity of tumor cells. It is currently little known about TGF-β1-promoted cell migration in hepatocellular carcinoma (HCC) cells, let alone its mechanism. In this study, we found that TGF-β1 lost its tumor-suppressive effects, but significantly stimulated cellmigration in SMMC-7721 human HCC cells. By FACS and Western blot analysis, we observed that TGF-β1 enhanced the expression of ct5131 integrin obviously, and subsequently stimulated cell adhesion onto fibronectin(Fn). Furthermore, we observed that TGF-β1 could also promote SMMC-7721 cells adhesion onto laminin (Ln).Our data also provided evidences that TGF-β1 induced epithelial-to-mesenchymal transformation (EMT) in SMMC-7721 cells. First, SMMC-7721 cells clearly switched to the spindle shape morphology after TGF-β1 treatment.Furthermore, TGF-β1 induced the down-regulation of E-cadherin and the nuclear translocation of β1-catenin. These results indicated that TGF-β1-promoted cell adhesion and TGF-β1-induced epithelial-to-mesenchymal transfor-mation might be both responsible for TGF-β1-enhanced cell migration.     

Can overexpression of TGF—β1 gene change the sex ratio in transgenic mice？

Mouse TGF-β1 gene was microinjected into male pronuclei of F2 hybrid fertilized eggs obtained by mating CSJLF1 and C57BL/6J inbred strains to generate transgenic mice with over-expressed TGF-β1 gene.The rate of founder production is 31% and Southern blot analysis of founder mice tail DNAs gave an integration efficiency of 33%.TGF-β1 gene could be stably integrated to the chromosomes of transgenic mice and transmitted to their progeny at a rate of 33% in the second generation.Dot blot analysis of tail RNA of some transgenic mice indicated a moderate expression of the transgene.The most interestin finding of the present work is the striking eviation from the normal male:female sex ratio in transgenic mice,with an average ratio of 6.7:1.The possible nature of the predominance of male sex in transgenic mice overexpressing TGF-β1 is discussed.     

Transforming Growth Factor-β1 Induces Transdifferentiation of Fibroblasts into Myofibroblasts in Hypoxic Pulmonary Vascular Remodeling

The muscularization of non-muscular pulmonary arterioles is an important pathological feature of hypoxic pulmonary vascular remodeling. However, the origin of the cells involved in this process is still not well understood. The present study was undertaken to test the hypothesis that transforming growth factor-β1 （TGF-β1） can induce transdifferentiation of fibroblasts into myofibroblasts, which might play a key role in the muscularization of non-muscular pulmonary arterioles. It was found that mean pulmonary arterial pressure increased significantly after 7 d of hypoxia. Pulmonary artery remodeling index and fight ventricular hypertrophy became evident after 14 d of hypoxia. The distribution of nonmuscular, partially muscular, and muscular vessels was significantly different after 7 d of hypoxia. Immunocytochemistry results demonstrated that the expression of α-smooth muscle actin was increased in intra-acinar pulmonary arteries with increasing hypoxic time. TGF-β1 mRNA expression in pulmonary arterial walls was increased significantly after 14 d of hypoxia, but showed no obvious changes after 3 or 7 d of hypoxia. In pulmonary tunica adventitia and tunica media, TGF-β1 protein staining was poorly positive in control rats, but was markedly enhanced after 3 d of hypoxia, reaching its peak after 7 d of hypoxia. The myofibroblast phenotype was confirmed by electron microscopy, which revealed microfilaments and a well-developed rough endoplasmic reticulum. Taken together, our results suggested that TGF-β1 induces transdifferentiation of fibroblasts into myofibroblasts, which is important in hypoxic pulmonary vascular remodeling.     

Enhanced Invasive and Metastatic Potential Induced by Transforming Growth Factor-β1 Might be Correlated with Glutathione-S-transferase-π, Cofilin and Heat Shock Protein 27 in SGC-7901 Gastric Cancer Cells

In our previous study, we found that the transforming growth factor （TGF）-β1 enhanced the metastatic and invasive potential of gastric cancer cells. Proteomics was employed in this study to further illustrate the underlying molecular mechanisms. After two-dimensional electrophoresis, image analysis, spot identification, protein identification and database analysis, three proteins, namely, glutathione-S-transferase-π （GST-π）, cofilin and heat shock protein 27 （HSP27）, were found to be up-regulated in TGF-β1 treated SGC-7901 cells. The findings were further confirmed by Western blot analysis. These results suggested that GST-π, cofilin and HSP27 might participate in enhanced invasive potential induced by TGF-β1.     

Transforming Growth Factor-β1 Induces Transdifferentiation of Fibroblasts into Myofibroblasts in Hyp0Xic Pulmonary Vascular Remodeling

The muscularization of non-muscular pulmonary arterioles is an important pathological featureof hypoxic pulmonary vascular remodeling.However,the origin of the cells involved in this process is stillnot well understood.The present study was undertaken to test the hypothesis that transforming growthfactor-β1 (TGF-β1) can induce transdifferentiation of fibroblasts into myofibroblasts,which might play akey role in the muscularization of non-muscular pulmonary arterioles.It was found that mean pulmonaryarterial pressure increased significantly after 7 d of hypoxia.Pulmonary artery remodeling index and rightventricular hypertrophy became evident after 14 d of hypoxia.The distribution of nonmuscular,partiallymuscular,and muscular vessels was significantly different after 7 d of hypoxia.Immunocytochemistryresults demonstrated that the expression of co-smooth muscle actin was increased in intra-acinar pulmonaryarteries with increasing hypoxic time.TGF-β1 mRNA expression in pulmonary arterial walls was increasedsignificantly after 14 d of hypoxia,but showed no obvious changes after 3 or 7 d of hypoxia.In pulmonarytunica adventitia and tunica media,TGF-β1 protein staining was poorly positive in control rats,but wasmarkedly enhanced after 3 d of hypoxia,reaching its peak after 7 d Of hypoxia.The myofibroblast phenotypewas confirmed by electron microscopy,which revealed microfilaments and a well-developed rough endo-plasmic reticulum.Taken together,our results suggested that TGF-β1 induces transdifferentiation of fibro-blasts into myofibroblasts,which is important in hypoxic pulmonary vascular remodeling.     

Role of glycosylation in TGF-β signaling and epithelial-to-mesenchymal transition in cancer

Glycosylation is a common posttranslational modification on membrane-associated and secreted proteins that is of pivotal importance for regulating cell functions.Aberrant glycosylation can lead to uncontrolled cell proliferation,cell-matrix interactions,migration and differentiation,and has been shown to be involved in cancer and other diseases.The epithelial-to-mesenchymal transition is a key step in the metastatic process by which cancer cells gain the ability to invade tissues and extravasate into the bloodstream.This cellular transformation process,which is associated by morphological change,loss of epithelial traits and gain of mesenchymal markers,is triggered by the secreted cytokine transforming growth factor-β(TGF-β).TGF-βbioactivity is carefully regulated,and its effects on cells are mediated by its receptors on the cell surface.In this review,we first provide a brief overview of major types of glycans,namely,N-glycans,O-glycans,glycosphingolipids and glycosaminoglycans that are involved in cancer progression.Thereafter,we summarize studies on how the glycosylation of TGF-βsignaling components regulates TGF-βsecretion,bioavailability and TGF-βreceptor function.Then,we review glycosylation changes associated with TGF-β-induced epithelial-to-mesenchymal transition in cancer.Identifying and understanding the mechanisms by which glycosylation affects TGF-βsignaling and downstream biological responses will facilitate the identification of glycans as biomarkers and enable novel therapeutic approaches.     

Effects of adeno-associated virus (AAV) of transforming growth factors β_1 and β_3 (TGFβ_(1,3)) on promoting synthesis of glycosaminoglycan and collagen type II of dedifferentiated nucleus pulposus (NP) cells

The effects of AAV-TGFβ1 and AAV-TGFβ3 on promoting synthesis of glycosaminoglycan and collagen type II of dedifferentiated rabbit lumbar disc NP cells were studied in this work. The rabbit lumbar disc NP cells were isolated and cultured. The earlier and later dedifferentiated NP cells were established by subculture. The AAV transfection efficiency to dedifferentiated NP cells was analyzed with AAV-EGFP in vitro. After dedifferentiated NP cells were transfected by AAV-TGFβ1 or AAV-TGFβ3, their biological effects on promoting synthesis of glycosaminoglycan or collagen type II were detected and compared by the methods of 35S incorporation or immunoblotting. The experimental results showed that AAV could transfect efficiently the earlier dedifferentiated NP cells, but its transfection rate was shown to be at a low level to the later dedifferentiated NP cells. Both AAV-TGFβ1 and AAV-TGFβ3 could promote the earlier dedifferentiated NP cells to synthesize glycosaminoglycan and collagen type II, and the effect of AAV-TGFβ1 was better than that of AAV-TGFβ3. For the later dedifferentiated NP cells, the AAV-TGFβ3 could promote their synthesis, but AAV-TGFβ1 could slightly inhibit their synthesis. Therefore, AAV-TGFβ1 and AAV-TGFβ3 could be used for the earlier dedifferentiated NP cells, and the TGFβ3 could be used as the objective gene for the later dedifferentiated NP cells.     

Proliferation and tenogenic differentiation of bone marrow mesenchymal stem cells in a porous collagen sponge scaffold

BACKGROUND Collagen is one of the most commonly used natural biomaterials for tendon tissue engineering.One of the possible practical ways to further enhance tendon repair is to combine a porous collagen sponge scaffold with a suitable growth factor or cytokine that has an inherent ability to promote the recruitment,proliferation,and tenogenic differentiation of cells.However,there is an incomplete understanding of which growth factors are sufficient and optimal for the tenogenic differentiation of rat bone marrow mesenchymal stem cells(BMSCs)in a collagen sponge-based 3D culture system.AIM To identify one or more ideal growth factors that benefit the proliferation and tenogenic differentiation of rat BMSCs in a porous collagen sponge scaffold.METHODS We constructed a 3D culture system based on a type I collagen sponge scaffold.The surface topography of the collagen sponge scaffold was observed by scanning electron microscopy.Primary BMSCs were isolated from Sprague-Dawley rats.Cell survival on the surfaces of the scaffolds with different growth factors was assessed by live/dead assay and CCK-8 assay.The mRNA and protein expression levels were confirmed by quantitative real-time polymerase chain reaction and Western blot,respectively.The deposited collagen was assessed by Sirius Red staining.RESULTS Transforming growth factorβ1(TGF-β1)showed great promise in the tenogenic differentiation of BMSCs compared to growth differentiation factor 7(GDF-7)and insulin-like growth factor 1(IGF-1)in both the 2D and 3D cultures,and the 3D culture enhanced the differentiation of BMSCs into tenocytes well beyond the level of induction in the 2D culture after TGF-β1 treatment.In the 2D culture,the proliferation of the BMSCs showed no significant changes compared to the control group after TGF-β1,IGF-1,or GDF-7 treatment.However,TGF-β1 and GDF-7 could increase the cell proliferation in the 3D culture.Strangely,we also found more dead cells in the BMSC-collagen sponge constructs that were treated with TGF-β1.Moreover,TGF-β1 promoted more collagen deposition in both the 2D and 3D cultures.CONCLUSION Collagen sponge-based 3D culture with TGF-β1 enhances the responsiveness of the proliferation and tenogenic differentiation of rat BMSCs.     

Effects of adeno-associated virus (AAV) of transforming growth factors β1 and β3 (TGFβ1,3) on promoting synthesis of glycosaminoglycan and collagen type II of de-differentiated nucleus pulposus (NP) cells

The effects of AAV-TGFβ1 and AAV-TGFβ3 on promoting synthesis of glycosaminoglycan and collagen type Ⅱ of dedifferentiated rabbit lumbar disc NP cells were studied in this work. The rabbit lumbar disc NP cells were isolated and cultured. The earlier and later dedifferentiated NP cells were established by subculture. The AAV transfection efficiency to dedifferentiated NP cells was analyzed with AAV-EGFP in vitro. After dedifferentiated NP cells were transfected by AAV-TGFβ1 or AAV-TGFβ3, their biological effects on promoting synthesis of glycosaminoglycan or collagen type Ⅱ were detected and compared by the methods of 35S incorporation or immunoblotting. The experimental results showed that AAV could transfect efficiently the earlier dedifferentiated NP cells, but its transfection rate was shown to be at a low level to the later dedifferentiated NP cells. Both AAV-TGFβ1 and AAV-TGFβ3 could promote the earlier dedifferentiated NP cells to synthesize glycosaminoglycan and collagen type Ⅱ, and the effect of AAV-TGFβ1 was better than that of AAV-TGFβ3. For the later dedifferentiated NP cells, the AAV-TGFβ3 could promote their synthesis, but AAV-TGFβ1 could slightly inhibit their synthesis. Therefore, AAV-TGFβ1 and AAV-TGFβ3 could be used for the earlier dedifferentiated NP cells, and the TGFβ3 could be used as the objective gene for the later dedifferentiated NP cells.     

Transforming growth factor-β1 suppresses serum deprivation-induced apop-tosis via activation of ERK1/ERK2 and the inhibition of p38 activity and ceramide production

In this report we studied the effects and mechanism of transforming growth factor-β1 (TGF-β1) on serum deprivation-induced cell apoptosis. Serum deprivation induces apoptosis, which is associated with an increase in intracellular ceramide level and with the activation of p38 mitogen-activated protein (MAP) kinase. Inhibition of p38 MAP kinase by SB203580 significantly reduced apoptosis induced by serum-deprivation. Treatment of cells with TGF-β1 stimulated cell proliferation and suppressed the serum deprivation-induced apoptotic response. The anti-apoptotic effect of TGF-β1 is correlated with its ability to inhibit the serum deprivation-induced activation of p38 MAP kinase and the increase in intracellular ceramide level. In     

TGF—β receptors in mouse ES—5 cells and their differentiated derivatives

By radioreceptor binding studies with iodinated TGF－β1,it has been shown that an undifferentiated ES-5 cell expresses approximately 3270 receptors with a dissociation constant Kd-130pM,but after the induction of differentiation by retinoic acid and dBcAMP,the receptor number of a differentiated RA-ES-5 cell was increased about 80% and the Kd was also increased to 370 pM.Furthermore,more direct evidence supporting the expression of TGF-β type I and type Ⅱ receptors in both ES-5 and RA-ES-5 cells has come from dot blot hybridization of cellular mRNA with cDNA probes for type I and type Ⅱ receptors.Meanwhile,mRNA expression level of types I and Ⅱ receptors in R-ES-5 cells were higher than that in ES-5 cells.Down-regulation of TGF-β receptors with a significant decrease in the rate of cell proliferation in both cells,was found by employing a pretreatment with neutralizing antibody to TGF-β1.The possible role of receptors for TGF-β in cell differentiation is discussed here.