Project description:Activated hepatic stellate cells/myofibroblasts induced by TGF-β within the hepatic microenvironment are crucial determinants for cell growth and liver metastasis of colorectal cancer. Notably, TGF-β receptor II serves as the initiator of the received signal, and its membrane localization, internalization, degradation, and trafficking play a decisive role in determining the duration and intensity of TGF-β/SMAD signaling. This study aimed to identify the proteins that interact with TGF-β receptor II and clarify the underlying mechanisms of myofibroblastic activation of HSCs.
Project description:In order to identify the genes that are regulated by TGF-beta in glioma, we serum starved two glioma cell lines, U373MG and U87MG, for 16h and we treated them with vehicle,100pM TGF-beta, 2uM inhibitor of the TGF-beta Receptor I(TbRI)(LY2109761), or both 100pM TGF-beta plus 2uM TbRI for 3h. Then, cell were collected and total RNA was extracted.
Project description:TGF-beta has an oncogenic response in glioblastoma and it is considered to be a therapeutic target. We evaluated the effect of TGF-beta inhibition in glioblastoma. Differential gene expression analysis of patient-derived primary cultured glioblastoma cells treated with the TGF-beta receptor inhibitor, LY2109761 11 patient-derived cell cultures from 11 patients were treated with 2microM of the TGF-beta receptor inhibitor, LY2109761, for 3 hours or left untreated and RNA was isolated and microarray analysis was performed
Project description:TGF-beta has an oncogenic response in glioblastoma and it is considered to be a therapeutic target. We evaluated the effect of TGF-beta inhibition in glioblastoma. Differential gene expression analysis of patient-derived primary cultured glioblastoma cells treated with the TGF-beta receptor inhibitor, LY2109761
Project description:The murine helminth parasite Heligmosomoides polygyrus expresses a family of modular proteins which, replicating the functional activity of the immunomodulatory cytokine TGF-β, have been named TGM (TGF-β Μimic). Multiple domains bind to different receptors, including TGF-β receptors TβRI (ALK5) and TβRII through domains 1-3, and prototypic family member TGM1 binds the cell surface co-receptor CD44 through domains 4-5. This allows TGM1 to induce T lymphocyte Foxp3 expression, characteristic of regulatory (Treg) cells, and to activate a range of TGF-β-responsive cell types. In contrast, a related protein, TGM4, targets a much more restricted cell repertoire, primarily acting on myeloid cells, with less potent effects on T cells and lacking activity on other TGF-β-responsive cell types. TGM4 binds avidly to myeloid cells by flow cytometry, and can outcompete TGM1 for cell binding. Analysis of receptor binding in comparison to TGM1 reveals a 10-fold higher affinity than TGM1 for TGFβR-I (TβRI), but a 100-fold lower affinity for TβRII through Domain 3. Consequently, TGM4 is more dependent on co-receptor binding; in addition to CD44, TGM4 also engages CD49d (Itga4) through Domains 1-3, as well as CD206 and Neuropilin-1 through Domains 4 and 5. TGM4 was found to effectively modulate macrophage populations, inhibiting lipopolysaccharide-driven inflammatory cytokine production and boosting interleukin (IL)-4-stimulated responses such as Arginase-1 in vitro and in vivo. These results reveal that the modular nature of TGMs has allowed the fine tuning of the binding affinities of the TβR- and co-receptor binding domains to establish cell specificity for TGF-β signalling in a manner that cannot be attained by the mammalian cytokine.
Project description:We looked at transcriptional changes in immortalized IMCD (inner medullary collecting duct cells) derived from Tgfbr2floxed mice, described in detail in manuscript PMID: 20576806
Project description:Analyze TGF-beta pathway transcriptional regulation in breast cancer stem cells with different responses upon TGF-beta pathway activation. Total RNA from four breast cell lines grown as mammospheres treated with recombinant TGF-beta or a TGF-beta receptor I inhibitor was used in the analysis.
Project description:Intestinal crypts isolated from Apcflox/flox; villin-CreERT mice were treated with Tamoxifen to induce the deletion of Apc. Tamoxifen-treated organoids were selected in the absence of Wnt agonists and then treated with TGF-beta. Total RNA obtained from Tamoxifen-treated, Apc-deleted intestinal organoids in the absence or presence of 3 ng/ml TGF-beta (18h).
Project description:Most available CRC cell lines have lost their TGF-beta response through the acquisition of mutations either in TGF-beta type II receptor (TGFBR2) or the intracellular mediator SMAD4 (Reviewed in Markowitz et al. 2002). To study the effect of TGF-beta in CRC epithelial cells, we restored wild-type TGFBR2 expression in the CRC cell line LS174T (LS). Ls174T cells are mutant for beta-catenin and therefore exhibit constitutive Wnt activity, which results in the expression of a genetic profile similar to that of intestinal epithelial progenitor cells (van de Wetering, Sancho et al. 2002). They thus reflect tumor cells at the initial stages of tumorogenesis. In addition, Ls174T cells bear mutations in the TGF-beta receptor II (TGFBR2) sequence at the BATRII locus (Grady and Markowitz 2002). This mutation gives rise to a non-functional truncated form of the receptor lacking the transmembrane domain and the serine-threonine kinase domain (Parsons, Myeroff et al. 1995). We generated inducible clones of Ls174T cells, where the expression of TGFBR2 could be controlled at will by the presence or absence of doxycycline (LSTGFBR2 cell line). Re-expression of wild-type receptor in Ls174T cells (LSTGFBR2) restored TGF-beta signalling resulting in strong phosphorylation of SMADs and activation of SMAD-binding reporter activity. Functionally, TGF-beta signalling in LSTGFBR2 resulted in a strong cytostatic response. By genome wide expression profiling using microarrays we investigated genes regulated by TGF-beta in this cell line. LsTGFBR2 inducible cells were generated by the Invitrogen T-Rex system following manufacturers instructions. LSTGFBR2 were seeded at 60% confluence in the presence or absence of Doxycycline and treated with TGF-M-NM-21 for 16 hours. Gene expression profiles were measured in duplicate using HG-U133 plus 2.0. We used RMA background correction, quantile normalization and RMA summarization (Gautier et al., 2004). A TGF-M-NM-2 response signature was obtained by selecting genes with limma P-value < 0.05 and at least two fold up-regulation in TGF-M-NM-2 treated LSTGFBR2 cells.
Project description:<p>The mechanisms by which macrophage metabolism is regulated and the effects of metabolism on diseases remain largely unknown. We show here that TGF-β regulates the glycolysis of macrophages independently of inflammatory cytokine production, and thus affects the survival in experimental sepsis. Specifically, TGF-β increased expression and activity of phosphofructokinase-1 liver type (PFKL) in macrophages and thus promoted their glycolysis during cell activation, yet paradoxically suppressed the production of proinflammatory cytokines in the same macrophages. The upregulation of glycolysis was mediated by a mTOR-c-MYC dependent pathway, whereas the inhibition of cytokines was ascribed to the activation of SMAD3 and a downregulated activation of the pro-inflammatory transcription factors AP-1, NFkB and STAT1. Importantly, in an LPS-induced endotoxemia and CLP-sepsis models, TGF-β enhancement of macrophage glycolysis led to a decreased survival in mice, which was associated with increased blood coagulation. Analysis of cohorts of patients with sepsis and covid-19 revealed that the expression of PFKL, TGF-β receptor TGFBRI and coagulation factor F13A1 in myeloid cells positively correlated with the progression of the disease. Thus, TGF-β is emerging as a critical cytokine regulating macrophage metabolism and could serve as a therapeutic target in patients with sepsis.</p>