Different Concentrations Of TGF-β1 Research Articles

Activation of extracellular signal-regulated kinase 1/2 and Sp1 may contribute to the expression of tissue inhibitor of metalloproteinases-1 induced by transforming growth factor-β1 in human pulmonary arterial smooth muscle cells

Background aimsTissue inhibitor of metalloproteinases-1 (TIMP-1) plays an important role in the development of pulmonary arterial hypertension. However, the molecular regulatory mechanisms of TIMP-1 in the pulmonary arteries are not fully understood, especially in human pulmonary arterial smooth muscle cells (HPASMCs). We investigated the signaling pathway involved in the regulation of TIMP-1 in HPASMCs induced by transforming growth factor (TGF)-β1. MethodsCultured HPASMCs were incubated with different concentrations of TGF-β1 (0–40 ng/mL) for 24 h or with 10 ng/mL TGF-β1 for different times (1–48 h). ResultsWestern blot, real-time polymerase chain reaction and enzyme-linked immunosorbent assay analyses showed that TGF-β1 enhanced the expression and secretion of TIMP-1 in a time-dependent and dose-dependent fashion. TGF-β1 could phosphorylate two of the three mitogen-activated protein kinases—extracellular signal-regulated kinase 1/2 (ERK1/2) and p38, but not c-Jun NH2-terminal kinase. Of these kinases, only the inhibition of ERK1/2 by U0126, which was a specific inhibitor of mitogen-activated protein kinase/ERK1/2, effectively blocked the TGF-β1-induced expression of TIMP-1. Mithramycin, an inhibitor of Sp1 transcription factor, also significantly inhibited the expression of TIMP-1. Additionally, electrophoretic mobility shift assay showed that TGF-β1 could up-regulate the DNA-binding activity of Sp1 and that U0126 and mithramycin could effectively inhibit these events. ConclusionsTGF-β1 could stimulate the expression and secretion of TIMP-1 in HPASMCs in a time-dependent and dose-dependent fashion, and ERK1/2 and Sp1 signaling pathways might be involved in these activities.

Premature senescence and cellular phenotype transformation of mesangial cells induced by TGF-B1

Background: Transforming growth factor-β1 (TGF-β1) is a polypeptide member of the transforming growth factor β superfamily of cytokines and performs many cellular functions. Its overexpression may lead to renal fibrosis. Aim: This study planed to investigate the effects of TGF-β1 on the cell cycle and phenotype of mesangial cells. Methods: Rat mesangial cells were cultured together with different concentrations (0, 1, 2, 5, and 10 ng/mL) of TGF-β1 for specified times from 0 min to 72 h. 0 ng/mL TGF-β1 and 0 min served as controls. Cell cycles were assessed by flow cytometry and α-smooth muscle actin expression (α-SMA) protein expression by western blot analysis. All data were presented as Mean ± SD. Statistical analysis was performed by using one-way analysis of variance and correlation analysis. Results were considered significant at p < 0.05. Results: After 15 min of co-culture with different concentrations of TGF-β1, the percentage of mesangial cells in G0/G1 phase was significantly elevated compared to the control (p < 0.05). 12 h co-culture induced cell hyperplasia, 24 h co-culture obvious up-regulation of α-SMA (p < 0.01) and one or two cells’ myofibroblast phenotype transition, and 36 h co-culture several cells’ phenotype transition. Correlation analysis prompted that the TGF-β1-induced premature aging was time-dependent (p < 0.01). Conclusion: TGF-β1 may induce mesangial cells’ premature senescence and myofibroblast-like phenotype transformation time-dependently, which may contribute to the development of early stage of glomerulosclerosis.

TGF-β1 promotes transition of mesothelial cells into fibroblast phenotype in response to peritoneal injury in a cell culture model

BackgroundPeritoneal adhesions are a clinical problem. A key to the understanding of peritoneal adhesions is to study the healing of mesothelial cells within the peritoneal cavity following surgery. Transforming growth factor beta (TGF-βs) affects this healing process. The aim of this study was to investigate the effects of different concentrations of TGF-β1 on the healing rate and healing properties of mesothelial cells. Materials and methodsHuman mesothelial cells from peritoneal fluid were collected, cultured and a mechanical wound was created. The restoration of the mesothelial surface with and without increasing concentrations of TGF-β1 was monitored. ResultsThe denuded area was restored within 24 h. The healing rate was most extensive between the first and second hour after the damage (61.9 ± 22.8 μm/h). No significant difference in healing rate were observed when increasing levels of TGF-β1 were used. However, higher concentrations of TGF-β1 increased cell size and the cells presented more fibroblast specific properties. Lower TGF-β1 concentrations increased the number of proliferating cells. ConclusionsThis study indicates the importance of high levels TGF-β1 in mesothelial cell healing, mainly by changing the actual healing properties of the cells. Elevated levels of TGF-β1 might promote mesothelial cell transition towards a more fibroblast-like appearance.

ACTIVATION OF ERK 1/2 AND SP1 MAY CONTRIBUTE TO THE EXPRESSION OF TISSUE INHIBITOR OF METALLOPROTEINASE-1 INDUCED BY TRANSFORMING GROWTH FACTOR-β1 IN HUMAN PULMONARY ARTERIAL SMOOTH MUSCLE CELLS

ObjectivesTissue inhibitor of metalloproteinases-1 (TIMP-1) is considered to play a key role in the development of pulmonary arterial hypertension (PAH). However, the molecular regulatory mechanisms of TIMP-1 in the pulmonary...

Biological activity of bovine milk on proliferation of human intestinal cells

To evaluate the bioactivity of bovine milk from different stages of lactation on human intestinal tissue, a human fetal small intestinal cell line was used as a model system. Milk samples representing six stages of lactation: days 1, 2-3, 6-7 and weeks 12 and 24 after parturition, 1 week before drying off, and milk-like secretion from two stages of the dry period: 7 weeks and 3-4 weeks before expected calving, were collected from 64 Holstein Friesian cows. The whey fraction of the milk or milk-like secretion was added to the culture medium in concentrations ranging from 0.078% to 10%. The growth-promoting activity of whey was measured by determining the incorporation of [3H]thymidine into DNA for the last 24 h of the culture period. Whey fractions from all six stages of lactation stimulated growth of intestinal cells. The growth-promoting activity of colostrum or milk significantly decreased within the first week after calving. The growth-promoting activity in mature milk increased gradually during lactation to reach a level significantly higher than that obtained with colostrum. The growth-promoting activity of whey from milk-like secretion collected after drying off was lower than that of colostrum. Whey from different stages of lactation contained significantly different concentrations of TGF-beta1 (0.5-27 ng/ml) and TGF-beta2 (12-1219 ng/ml). However, neither the differences in TGF-beta1 and TGF-beta2, nor the differences in IGF-I and IGF-binding proteins could fully explain the differences in growth-promoting activity of colostrums or milk from different stages of lactation, suggesting that other factors were also involved. The present study showed that bovine milk contained a number of biologically active components that affected growth and development of human intestinal tissue. The results showed that the growth-promoting activity of colostrum and milk was dependent on the stage of lactation in accordance with previous results obtained with mammary epithelial cells. The changes in growth-promoting activity with stage of lactation were probably related to changes in concentrations of several growth factors.

TGF-β1 Affects the Proliferation of Normal Human Bone Marrow Fibroblasts and Its Expresssion of Smad3 and Smad7 mRNA.

Current drug therapy in myelofibrosis with myeloid metaplasia (MMM) still can not get great progress, the development of the myelofibrosis eventually causes bone marrow failure. The histopathology of MMM patients showed that the bone marrow tissue were full of marrow fibroblasts and an extensive deposition of collagens. Further researches suggested that the proliferation of the fibroblasts was controlled by many factors. One of the most important factor was TGF-β1, and its effects largely depended on Smad pathway. In order to understand the effects of TGF-β1 on the proliferation of marrow fibroblasts, we stimulated the normal human bone marrow fibroblasts with different concentrations of TGF-β1 in the current study. The proliferation test with direct cell counting and MTT assay revealed that different concentrations of TGF-β1 had various effects and it could get the maximal proliferation effect at 0.1ng/ml. Then we detected the Smad3 mRNA and Smad7 mRNA in one normal human bone marrow fibroblasts with RT-PCT methods at above concentration (0.1ng/ml). We found that the expression level of Smad3 mRNA decreased with the time, and that the expression level of Smad7 mRNA reached the highest at 30 min after stimulation by TGF-β1, then it began to reduce. These results suggested that TGF-β1 activate the Smads pathway in normal human marrow fibroblasts, and its proliferation effect require the activation of this pathway. We also noticed that the changes of the smad3 mRNA and smad7 mRNA were similar in skin fibroblasts stimulated by TGF-β1. These changes were considered as a feedback modulation mechanism. Whether the over-proliferation of marrow fibroblasts in MMM patients is correlated with the imbalance of this feedback warrants further research. It may provide a more specific and targeted therapy for blocking myelofibrosis. [Display omitted]