Project description:Studies in experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS), have shown that regulatory B cells modulate the course of the disease via the production of suppressive cytokines. While data indicate a role for transforming growth factor (TGF)-β1 expression in regulatory B cell functions, this mechanism has not yet been tested in autoimmune neuroinflammation. Transgenic mice deficient for TGF-β1 expression in B cells (B-TGF-β1-/-) were tested in EAE induced by recombinant mouse myelin oligodendrocyte glycoprotein (rmMOG). In this model, B-TGF-β1-/- mice showed an earlier onset of neurologic impairment compared to their littermate controls. Exacerbated EAE susceptibility in B-TGF-β1-/- mice was associated with augmented CNS T helper (Th)1/17 responses. Moreover, selective B cell TGF-β1-deficiency increased the frequencies and activation of myeloid dendritic cells, potent professional antigen-presenting cells (APCs), suggesting that B cell-derived TGF-β1 can constrain Th1/17 responses through inhibition of APC activity. Collectively our data suggest that B cells can down-regulate the function of APCs, and in turn encephalitogenic Th1/17 responses, via TGF-β1, findings that may be relevant to B cell-targeted therapies.
Project description:Treatment for musculoskeletal fibromatosis remains challenging. Surgical excision for fibromatosis is the standard therapy but recurrence remains high. Corticosteroids, an anti-fibrogenic compound, have been used to treat early stage palmar fibromatosis, but the mechanism is unknown. We investigated the inhibitory mechanism effect of corticosteroids in the murine model of fibromatosis nodule as well as in cultured FSCs. Quantitative reverse transcription/polymerase chain reaction (PCR) analysis and immunofluorescence (IF) staining for markers of myofibroblasts (α-smooth muscle actin and type III collagen) were used to examine the effect of dexamethasone on myofibroblasic differentiation of FSCs both in vitro and in vivo. Transforming growth factor-β1 (TGF-β1) signaling and its downstream targets were examined using western blot analysis. TGF-β1 expression in FSCs before and after dexamethasone treatment was compared. In addition, inhibition of TGF-β1 expression was examined using RNA interference (RNAi) on FSCs, both in vitro and in vivo. Treating FSCs with dexamethasone inhibited FSCs' myofibroblastic differentiation in vitro. Treating FSCs with dexamethasone before or after implantation further inhibited formation of fibromatosis nodules. Dexamethasone suppressed expression of TGF-β1 and pSmad2/3 by FSCs in vitro. TGF-β1 knockdown FSCs showed reducing myofibroblastic differentiation both in vitro and in vivo. Finally, addition of TGF-β1 abolished dexamethasone-mediated inhibition of myofibroblastic differentiation. Dexamethasone inhibits the myofibroblastic differentiated potential of FSCs both in vitro and in vivo through inhibition of TGF-β1 expression in FSCs. TGF-β1 plays a key role in myofibroblastic differentiation.
Project description:Metformin is a widely used first-line antidiabetic drug that has been shown to protect against a variety of specific diseases in addition to diabetes, including cardiovascular disorders, polycystic ovary syndrome, and cancer. However, the precise mechanisms underlying the diverse therapeutic effects of metformin remain elusive. Here, we report that transforming growth factor-β1 (TGF-β1), which is involved in the pathogenesis of numerous diseases, is a novel target of metformin. Using a surface plasmon resonance-based assay, we identified the direct binding of metformin to TGF-β1 and found that metformin inhibits [(125)I]-TGF-β1 binding to its receptor. Furthermore, based on molecular docking and molecular dynamics simulations, metformin was predicted to interact with TGF-β1 at its receptor-binding domain. Single-molecule force spectroscopy revealed that metformin reduces the binding probability but not the binding force of TGF-β1 to its type II receptor. Consequently, metformin suppresses type II TGF-β1 receptor dimerization upon exposure to TGF-β1, which is essential for downstream signal transduction. Thus, our results indicate that metformin is a novel TGF-β suppressor with therapeutic potential for numerous diseases in which TGF-β1 hyperfunction is indicated.
Project description:Cartilage oligomeric matrix protein (COMP) is an important non-collagenous cartilage protein that is essential for the structural integrity of the cartilage extracellular matrix. The repeated modular structure of COMP allows it to "bridge" and assemble multiple cartilage extracellular matrix components such as collagens, matrilins, and proteoglycans. With its modular structure, COMP also has the potential to act as a scaffold for growth factors, thereby affecting how and when the growth factors are presented to cell-surface receptors. However, it is not known whether COMP binds growth factors. We studied the binding interaction between COMP and TGF-β1 in vitro and determined the effect of COMP on TGF-β1-induced signal transduction in reporter cell lines and primary cells. Our results demonstrate that mature COMP protein binds to multiple TGF-β1 molecules and that the peak binding occurs at slightly acidic pH. These interactions were confirmed by dual polarization interferometry and visualized by rotary shadow electron microscopy. There is cation-independent binding of TGF-β1 to the C-terminal domain of COMP. In the presence of manganese, an additional TGF-β-binding site is present in the TSP3 repeats of COMP. Finally, we show that COMP-bound TGF-β1 causes increased TGF-β1-dependent transcription. We conclude that TGF-β1 binds to COMP and that TGF-β1 bound to COMP has enhanced bioactivity.
Project description:In many tumors, CD73 (NT5E), a rate-limiting enzyme in adenosine biosynthesis, is upregulated by TGF-β and drives tumor progression. Conversely, CD73 is downregulated in endometrial carcinomas (EC) despite a TGF-β-rich environment. Through gene expression analyses of normal endometrium samples of the uterine cancer TCGA data set and genetic and pharmacological studies, we discovered CD73 loss shifts TGF-β1 from tumor suppressor to promoter in EC. TGF-β1 upregulated CD73 and epithelial integrity in vivo in the normal endometrium and in vitro in early stage EC cells. With loss of CD73, TGF-β1-mediated epithelial integrity was abrogated. EC cells developed TGF-β1-mediated stress fibers and macromolecule permeability, migration, and invasion increased. In human tumors, CD73 is downregulated in deeply invasive stage I EC. Consistent with shifting TGF-β1 activity, CD73 loss increased TGF-β1-mediated canonical signaling and upregulated cyclin D1 (CCND1) and downregulated p21 expression. This shift was clinically relevant, as CD73Low/CCND1High expression associated with poor tumor differentiation, increased myometrial and lymphatic/vascular space invasion, and patient death. Further loss of CD73 in CD73Low expressing advanced stage EC cells increased TGF-β-mediated stress fibers, signaling, and invasiveness, whereby adenosine A1 receptor agonist, CPA, dampened TGF-β-mediated invasion. These data identify CD73 loss as essential for shifting TGF-β activity in EC.
Project description:Transforming growth factor (TGF)-β1, a main profibrogenic cytokine in the progression of idiopathic pulmonary fibrosis (IPF), induces differentiation of pulmonary fibroblasts to myofibroblasts that produce high levels of collagen, leading to concomitantly loss of lung elasticity and function. Recent studies implicate the importance of microRNAs (miRNAs) in IPF but their regulation and individual pathological roles remain largely unknown. We used both RNA sequencing and quantitative RT-PCR strategies to systematically study TGF-β1-induced alternations of miRNAs in human lung fibroblasts (HFL). Our data show that miR-133a was significantly upregulated by TGF-β1 in a time- and concentration-dependent manner. Surprisingly, miR-133a inhibits TGF-β1-induced myofibroblast differentiation whereas miR-133a inhibitor enhances TGF-β1-induced myofibroblast differentiation. Interestingly, quantitative proteomics analysis indicates that miR-133a attenuates myofibroblast differentiation via targeting multiple components of TGF-β1 profibrogenic pathways. Western blot analysis confirmed that miR-133a down-regulates TGF-β1-induced expression of classic myofibroblast differentiation markers such as ɑ-smooth muscle actin (ɑ-SMA), connective tissue growth factor (CTGF) and collagens. miRNA Target Searcher analysis and luciferase reporter assays indicate that TGF-β receptor 1, CTGF and collagen type 1-alpha1 (Col1a1) are direct targets of miR-133a. More importantly, miR-133a gene transferred into lung tissues ameliorated bleomycin-induced pulmonary fibrosis in mice. Together, our study identified TGF-β1-induced miR-133a as an anti-fibrotic factor. It functions as a feed-back negative regulator of TGF-β1 profibrogenic pathways. Thus, manipulations of miR-133a expression may provide a new therapeutic strategy to halt and perhaps even partially reverse the progression of IPF.
Project description:Connective tissue growth factor (CTGF) associated with transforming growth factor-β (TGF-β) play a pivotal role in the pathophysiology of many fibrotic disorders. However, it is not clear whether this interaction also takes place in GO. In this study, we investigated the role of CTGF in TGF-β-induced extracellular matrix production and myofibroblast transdifferentiation in Graves' orbital fibroblasts. By Western blot analysis, we demonstrated that TGF-β1 induced the expression of CTGF, fibronectin, and alpha-smooth muscle actin (α-SMA) in Graves' orbital fibroblasts. In addition, the protein levels of fibronectin and α-SMA in Graves' orbital fibroblasts were also increased after treatment with a recombinant human protein CTGF (rhCTGF). Moreover, we transfected the orbital fibroblasts with a small hairpin RNA of CTGF gene (shCTGF) to knockdown the expression levels of CTGF, which showed that knockdown of CTGF significantly diminished TGF-β1-induced expression of CTGF, fibronectin and α-SMA proteins in Graves' orbital fibroblasts. Furthermore, the addition of rhCTGF to the shCTGF-transfected orbital fibroblasts could restore TGF-β1-induced expression of fibronectin and α-SMA proteins. Our findings demonstrate that CTGF is an essential downstream mediator for TGF-β1-induced extracellular matrix production and myofibroblast transdifferentiation in Graves' orbital fibroblasts and thus may provide with a potential therapeutic target for treatment of GO.
Project description:In rodent models of depression, (R)-ketamine has greater potency and longer-lasting antidepressant effects than (S)-ketamine; however, the precise molecular mechanisms underlying the antidepressant actions of (R)-ketamine remain unknown. Using RNA-sequencing analysis, we identified novel molecular targets that contribute to the different antidepressant effects of the two enantiomers. Either (R)-ketamine (10 mg/kg) or (S)-ketamine (10 mg/kg) was administered to susceptible mice after chronic social defeat stress (CSDS). RNA-sequencing analysis of prefrontal cortex (PFC) and subsequent GSEA (gene set enrichment analysis) revealed that transforming growth factor (TGF)-β signaling might contribute to the different antidepressant effects of the two enantiomers. (R)-ketamine, but not (S)-ketamine, ameliorated the reduced expressions of Tgfb1 and its receptors (Tgfbr1 and Tgfbr2) in the PFC and hippocampus of CSDS susceptible mice. Either pharmacological inhibitors (i.e., RepSox and SB431542) or neutralizing antibody of TGF-β1 blocked the antidepressant effects of (R)-ketamine in CSDS susceptible mice. Moreover, depletion of microglia by the colony-stimulating factor 1 receptor (CSF1R) inhibitor PLX3397 blocked the antidepressant effects of (R)-ketamine in CSDS susceptible mice. Similar to (R)-ketamine, the recombinant TGF-β1 elicited rapid and long-lasting antidepressant effects in animal models of depression. Our data implicate a novel microglial TGF-β1-dependent mechanism underlying the antidepressant effects of (R)-ketamine in rodents with depression-like phenotype. Moreover, TGF-β1 and its receptor agonists would likely constitute a novel rapid-acting and sustained antidepressant in humans.
Project description:Previously we reported that TGF-β1-induced growth suppression was associated with a decrease in mutant p53 levels in B-cell lymphoma cells. The goal of the present study was to understand the mechanism involved in TGF-β1-mediated down-regulation of mutant p53. In RL and CA46, two B-cell lymphoma cell lines, TGF-β1 treatment caused down-regulation of E2F-1 transcription factor resulting in the down-regulation of both p14(ARF) and mutant p53, leading to growth arrest. Experimental overexpression of E2F-1 increased p14(ARF) level and blocked TGF-β1-induced down-regulation of p14(ARF). Overexpression of p14(ARF) blocked the down-regulation of mutant p53 and prevented growth arrest. p14(ARF) also attenuated TGF-β1-induced p21(Cip1/WAF1) induction, which was reversible by p53 siRNA, indicating the involvement of mutant p53 in controlling the TGF-β1-induced expression of p21(Cip1/WAF1). The interaction observed between phospho-Smad2 and mutant p53 in the nucleus could be the mechanism responsible for blocking the growth-suppressive effects of TGF-β1. In RL cells, p14(ARF) is present in a trimer consisting of mutant p53-Mdm2-p14(ARF) and in a dimer consisting of Mdm2-p14(ARF). Because it is known that Mdm2 can degrade p53, it is possible that, in its trimeric form, p14(ARF) is able to stabilize mutant p53 by inhibiting Mdm2. In its dimeric form, p14(ARF) may be sequestering Mdm2, limiting its ability to degrade p53. Collectively, these data demonstrate a unique mechanism in which the inhibition of TGF-β1-mediated growth suppression by mutant p53 can be reversed by the down-regulation of its stabilizing protein p14(ARF). This work suggests that the high levels of p14(ARF) often found in tumor cells could be a potential therapeutic target.
Project description:BackgroundIncreased Galectin 3-binding protein (LGALS3BP) serum levels have been used to assess hepatic fibrosis stages and the severity of hepatocellular carcinoma (HCC). Considering the crucial role of transforming growth factor-β1 (TGF-β1) in the emergence of these diseases, the present study tested the hypothesis that LGALS3BP regulates the TGF-β1 signaling pathway.MethodsThe expression levels of LGALS3BP and TGFB1 were analyzed in patients with metabolic dysfunction-associated steatohepatitis (MASH) and HCC. Multiple omics techniques, such as RNA-sequencing, transposase-accessible chromatin-sequencing assay, and liquid chromatography-tandem mass spectrometry proteomics, were used to identify the regulatory mechanisms for the LGALS3BP-TGF-β1 axis. The effects of altered TGF-β1 signaling by LGALS3BP were investigated in conditional LGALS3BP-knockin and LGALS3BP-knockout mice.ResultsIn patients with MASH and HCC, the levels of LGALS3BP and TGFB1 exhibited positive correlations. Stimulation of LGALS3BP by the inflammatory cytokine interferon α in HCC cells or ectopic overexpression of LGALS3BP in hepatocytes promoted the expression levels of TGFB1. Aggravated fibrosis was observed in the livers of hepatocyte-specific LGALS3BP-knockin mice, with increased TGFB1 levels. LGALS3BP directly bound to and assembled integrin αV, an integral mediator required for releasing active TGF-β1 from extracellular latent complex with the rearranged F-actin cytoskeleton. The released TGF-β1 activated JunB transcription factor, which in turn promoted the TGF-β1 positive feedback loop. LGALS3BP deletion in the hepatocytes downregulated TGF-β1 signaling and CCl4 induced fibrosis. Moreover, LGALS3BP depletion hindered hepatocarcinogenesis by limiting the availability of fibrogenic TGF-β1.ConclusionLGALS3BP plays a crucial role in hepatic fibrosis and carcinogenesis by controlling the TGF-β1 signaling pathway, making it a promising therapeutic target in TGF-β1-related diseases.