Effect Of TGFB1 Research Articles

The Effects of Tgfb1 and Csf3 on Chondrogenic Differentiation of iPS Cells in 2D and 3D Culture Environment.

Mesenchymal stem (MS) cells, embryonic stem (ES) cells, and induced pluripotent stem (iPS) cells are known for their ability to differentiate into different lineages, including chondrocytes in culture. However, the existing protocol for chondrocyte differentiation is time consuming and labor intensive. To improve and simplify the differentiation strategy, we have explored the effects of interactions between growth factors (transforming growth factor β1 (Tgfb1) and colony stimulating factor 3 (Csf3), and culture environments (2D monolayer and 3D nanofiber scaffold) on chondrogenic differentiation. For this, we have examined cell morphologies, proliferation rates, viability, and gene expression profiles, and characterized the cartilaginous matrix formed in the chondrogenic cultures under different treatment regimens. Our data show that 3D cultures support higher proliferation rate than the 2D cultures. Tgfb1 promotes cell proliferation and viability in both types of culture, whereas Csf3 shows positive effects only in 3D cultures. Interestingly, our results indicate that the combined treatments of Tgfb1 and Csf3 do not affect cell proliferation and viability. The expression of cartilaginous matrix in different treatment groups indicates the presence of chondrocytes. We found that, at the end of differentiation stage 1, pluripotent markers were downregulated, while the mesodermal marker was upregulated. However, the expression of chondrogenic markers (col2a1 and aggrecan) was upregulated only in the 3D cultures. Here, we report an efficient, scalable, and convenient protocol for chondrogenic differentiation of iPS cells, and our data suggest that a 3D culture environment, combined with tgfb1 and csf3 treatment, promotes the chondrogenic differentiation.

Chronic Hyperglycaemia Induced Alterations of Hepatic Stellate Cells Differ from the Effect of TGFB1, and Point toward Metabolic Stress.

The deleterious effect of hyperglycemia on the biology of the liver is supported by clinical evidence. It can promote the development of fatty liver, liver fibrosis, even liver cancer as complication of diabetes mellitus. As liver fibrosis is the consequence of hepatic stellate cell (HSC) activation, the questions were addressed whether alterations induced by high glucose concentration are directly related to TGFB1 effect, or other mechanisms are activated. In order to obtain information on the response of HSC for high glucose, LX-2 cells (an immortalized human HSC cell lineage) were cultured in 15.3mM glucose containing medium for 21days. The effect of glucose was compared to that of TGFB1. Our data revealed that chronic exposure of high glucose concentration initiated profound alteration of LX-2 cells and the effect is different from those observed upon interaction with TGFB1. Whereas TGFB1 induced the production of extracellular matrix proteins, high glucose exposure resulted in decreased MMP2 activity, retardation of type I collagen in the endoplasmic reticulum, with decreased pS6 expression, pointing to development of endoplasmic stress and sequestration of p21CIP1/WAF1 in the cytoplasm which can promote the proliferation of LX2 cells.

The Effect of TGFB1 and CD14 Gene Polymorphisms on the Clinical Findings of Cystic Fibrosis in Turkish Patients

The Effect of TGFB1 and CD14 Gene Polymorphisms on the Clinical Findings of Cystic Fibrosis in Turkish Patients

The Effect of TGFB1 and CD14 Gene Polymorphisms on the Clinical Findings of Cystic Fibrosis in Turkish Patients

The Effect of TGFB1 and CD14 Gene Polymorphisms on the Clinical Findings of Cystic Fibrosis in Turkish Patients

Abstract PR005: TGF-beta1 primed myeloid derived suppressor cells decrease tumor growth and lose their ability to inhibit T cell proliferation via iNOS downregulation

Abstract Background: Myeloid derived suppressor cells or MDSC are a heterogeneous population of bone marrow-derived cells that consist of myeloid progenitor and immature. Cancer-induced pro-inflammatory signals recruit MDSC from the bone marrow and maintain them in an undifferentiated state. MDSCs inhibit T cell proliferation via up regulation of iNOS and ROS thereby suppressing antitumor immune responses. MDSC have also been shown to promote tumor growth by stimulating angiogenesis and other mechanisms. TGF-b1 is a pleiotropic cytokine abundantly expressed in the tumor microenvironment with diverse effects on myeloid, lymphoid and tumor cells. The aim of this study is to determine the effect of TGF-b1 in the generation and function of MDSC, including its effects on T cell proliferation and tumor growth. Methods: Ex vivo MDSC generation: Bone marrow progenitor cells were derived from WT C57bl/6 mice and co-cultured with MTEC (mouse tonsil epithelial cells transformed with HPV16 E6+E7 oncogenes and H-Ras ) tumor supernatants in the presence or absence of TGF-β1 for 5 days at 370C. Cells were then harvested, processed into single cell suspensions, and stained for MDSC surface markers, DAF-DA (to determine nitric oxide levels),iNOS and other functional markers analysis was performed by flow cytometry (FACS). MDSC functional assay: A) T cell proliferation assay MDSCs were generated in the presence or absence of TGF-β1 with supernatants from MTEC cells and co-cultured with CFSE labeled T cells activated with anti CD3 and anti CD28 antibodies. T cell proliferation was measured by using CFSE dilution, which was analyzed by flow cytometry. B) Effect of MDSC on tumor growth Control and TGF-β1 conditioned MDSC were co-cultured with MTEC tumor (grown as spheroids) for 72 hrs at the end of which histological sections of the spheroids were prepared and analyzed for tumor proliferation by Ki-67 staining. Sorted MDSCs were also co-cultured with T-hep3 cells grown in a monolayer (human oral cancer line) and tumor growth was determined by flow cytometry. Results: While control MDSC suppressed T cell proliferation in a dose-dependent fashion, we observed that TGF-β1 primed MDSCs lost the ability to inhibit T cell proliferation. Further, TGF-β1 primed MDSC inhibited tumor growth in an ex vivo co-culture system. Histological sections of tumor spheroid / MDSC co-cultures revealed diminshed ki-67 expression in spheroids cultured with TGF-B1 conditioned MDSC compared to control. Upon further examining the cellular mechanism, it was seen that TGF-β1 treated MDSCs down regulate iNOS expression and produced decreased amounts of nitric oxide compared to their control counterparts, without altering the expression of other MDSC functional markers like arginase, PD-1 and PD-L1. Conclusions: We conclude that TGF-β1 reprograms MDSC via an iNOS/NO dependent mechanism to a) T cell suppressive capacity and b) inhibit tumor cell growth. These observations have a direct translational implication wherein the inherent pro-tumor nature of MDSCs could potentially be reprogrammed with TGF-β1 and directed toward the tumor thereby suppressing tumor growth. Citation Format: Padmini Jayaraman, Falguni Parikh, Rosemarie Krupar, Robin Parihar, Indu Varier, Andrew Sikora. TGF-beta1 primed myeloid derived suppressor cells decrease tumor growth and lose their ability to inhibit T cell proliferation via iNOS downregulation. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr PR005.

Expression and function of placental uric acid transporter GLUT9

Expression and function of placental uric acid transporter GLUT9

Association of gene variants in the TGF-beta signalling pathways with invasive breast cancer risk

Many studies of normal cells in vitro, of transgenic mouse models and of somatic mutations in human cancers have provided evidence that the cytokine transforming growth factor beta (TGFβ) acts as a suppressor of primary tumour initiation. However, studies of transformed cells in vitro and of mouse models have also implicated TGFβ as a promoter of the later stages of tumour development. A hypothesis that has been proposed to account for this dual action is that TGFβ acts as a tumour suppressor through the ubiquitous ALK5 receptor signalling via SMAD2 and SMAD3 to inhibit proliferation of primary tumour cells, but acts subsequently through the endothelial-specific ALK1 receptor via SMAD1 and SMAD5 to promote angiogenesis, which is required for tumour progression [1]. In a recent meta-analysis we showed that a single nucleotide polymorphism (SNP) generating a leucine to proline substitution in the signal peptide of the TGFB1 protein is associated with an increase in the risk of invasive breast cancer (OR per additional proline allele = 1.08 (95% CI = 1.04 to 1.11), Ptrend = 2.8 × 10-5) [2]. We have also reported that this SNP increases the amount of TGFB1 protein secreted in vitro by threefold [3]. These data suggest that higher levels of TGFB1 may promote the invasive breast tumour phenotype. To determine the effect of host TGFB1 levels in an in vivo model, Tgfb1+/- and Tgfb1+/+ mice have been compared in which the mice carry one or two TGFB1 alleles with the ancestral SNP form encoding proline in the signal peptide. These studies have revealed major effects of TGFB1 in controlling the site of metastatic seeding and the number of metastases that develop. The TGFB1 SNP association with breast cancer suggested that other genes in the TGFβ signalling pathways might be associated with altered risk. We have conducted association studies with SNPs in 16 further genes encoding proteins directly implicated in TGFβ signalling. LTBP1, LTBP2, LTBP4, TGFB1, TGFB2, TGFB3, ALK1, ALK5, TGFBR2, Endoglin, SMAD1, SMAD2, SMAD3, SMAD4, SMAD5, SMAD6 and SMAD7 were analysed. A comprehensive SNP tagging approach was used to select variants for genotyping in a staged study design using up to 6,900 cases and 6,900 controls, all collected from the East Anglia region of the UK (>98% of northwestern European ancestry). From 1,254 common SNPs (minor allele frequency >0.05) in these genes identified from the International HapMap project data, we defined and genotyped 354 tagging SNPs in the East Anglia cases and controls. Statistically significant associations were followed up by genotyping in a Polish set of 2,215 cases and 2,374 controls. Meta-analysis of these results identified associations with cancer susceptibility of a variant in ALK5 (OR per additional rare G allele = 0.88 (95% CI = 0.81 to 0.95), Ptrend = 0.001) and in TGFBR2 (OR per additional rare G allele = 0.96 (95% CI = 0.92 to 1.00), Ptrend = 0.039). Data from two genome-wide studies have been examined to search further for associations in these genes. The haplotype risks and interactions between two or more loci have been investigated and survival analyses have been conducted. From this comprehensive study we have identified tagging SNPs in two TGFβ receptor genes that are significantly associated with risk of invasive breast cancer. Identification of the causative SNPs within the LD blocks tagged by the two SNPs and their effects on signalling via the ALK1 and ALK5 pathways remain to be determined.

207. Effect of TGFB1 on mammary gland development is dependent on cellular source of gene expression

The cytokine TGFB1 is implicated in development of the mammary gland through regulation of epithelial cell proliferation and differentiation during puberty and pregnancy. We have compared mammary gland morphogenesis in virgin Tgfb1+/+, Tgfb1+/− and Tgfb1−/− mice, and transplanted Tgfb1+/+ and Tgfb1−/− epithelium to determine the impact of TGFB1 deficiency on development. When mammary gland tissue was evaluated respective to timing of puberty, invasion through the mammary fat pad of the ductal epithelium progressed similarly irrespective of genotype, albeit fewer terminal end buds were observed in mammary glands from Tgfb1−/− mice. The terminal end buds appeared morphologically normal, and a comparable amount of epithelial proliferation was evident. However, when transplanted into wildtype recipients, Tgfb1−/− epithelium showed accelerated invasion compared with Tgfb1+/+ epithelium. This suggests that the normal rate of ductal extension in Tgfb1−/− null mutant mice is the net result of impaired endocrine or paracrine support acting to limit the consequences of unrestrained epithelial growth. By adulthood, mammary glands in cycling virgin Tgfb1−/− mice were morphologically similar to those in Tgfb1+/+ and Tgfb1+/− animals, a normal branching pattern was observed, and the tissue differentiated into early alveolar structures in the diestrus phase of the ovarian cycle. Transplanted mammary gland epithelium showed a similar extent of ductal branching and evidence of secretory differentiation of luminal cells in pregnancy. These results reveal two opposing actions of TGFB1 during pubertal mammary gland morphogenesis, dependent on the cellular source of gene expression. When expressed in the epithelium, TGFB1 inhibits epithelial ductal growth, when expressed systemically, TGFB1 promotes epithelial ductal growth.

Transforming growth factor β (TGF-β) promotes invasiveness of ras-transformed rat intestinal epithelial (RIE) cells

Methods: An inducible form of H-Ras (V12) was transfected into the RIE cells (RIE:iRas) and was inducible by treatment with isopropyl-1thio-b-D-galactopyranoside (IPTG). Separately, The RIE:iRas cells were also transfected with a dominant negative form of the TbRII (DNIIR). The effect of TGF-b1 on cell invasiveness was examined in a Boyden chamber with type I collagen-coated transwell inserts. Cells were seeded on the inserts after the treatment with IPTG for 0168 h and stimulated with 5 ng/ml TGF-b1 for 24 h. TbRII expression was assessed by immunoblotting and immunofluorescence.

Cytoreductive therapy of multidrug-resistant hepatocellular carcinoma: negative regulation of growth using combination differentiation therapy.

In vitro studies of the mouse erythroleukemia cell system have identified at least 300 agents capable of inducing differentiation by mechanisms that remain to be elucidated. We have recently begun to examine recombinant cytokines as possible agents in inducing differentiation of tumor cells, specifically, malignant cells resistant to cytotoxic drugs. One such cytokine, transforming growth factor-beta (TGF-B1), is a multifunctional peptide that exists in at least five different isoforms in vertebrate species. Recently, there has been a great deal of interest in the role of TGF-B1 as an important multifunctional growth regulator that induces cells of mesenchymal origin to divide while inhibiting the growth of nontransformed epithelial cells. In this study, we combined the effects of the differentiation agent hexamethylene bisacetamide and the inhibiting effects of TGF-B1 on a multidrug-resistant human liver hepatocellular carcinoma and demonstrated the synergistic interaction of these two agents; this synergy resulted in a cell death rate of 80%. These data support the concept of programmed cell death and suggest that drug-resistant tumor cells may be susceptible to the combination of cytokines and differentiating agents.