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TGF-β receptor I inhibitor may restrict the induction of EMT in inflamed intestinal epithelial cells.

ABSTRACT: Despite the extensive body of research, understanding the exact molecular mechanisms governing inflammatory bowel diseases (IBDs) still demands further investigation. Transforming growth factor-β1 (TGF-β1) signaling possesses a multifacial effect on a broad range of context-dependent cellular responses. However, long-term TGF-β1 activity may trigger epithelial-mesenchymal transition (EMT), followed by fibrosis. This study aimed to determine the role of epithelial TGF-β1 signaling in inflammatory bowel disease (IBD) pathogenesis. The expression of TGF-β1 signaling components and EMT-related and epithelial tight junction markers was examined in IBD patients (n = 60) as well as LPS-induced Caco-2/RAW264.7 co-culture model using quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting, and immunofluorescence staining. Furthermore, the effect of A83-01, as a TGF-β receptor I (TβRI) inhibitor, on the inflamed epithelial cells was evaluated in vitro. To evaluate the cytotoxic effects of the TβRI inhibitor, a cell viability assay was performed by the MTS method. Considering the activation of canonical and non-canonical TGF-β1 signaling pathways in IBD patients, expression results indicated that administering A83-01 in inflamed Caco-2 cells substantially blocked the expression level of TGF-β1, SMAD4, and PI3K and the phosphorylation of p-SMAD2/3, p-AKT, and p-RPS6 as well as prevented downregulation of LncGAS5 and LncCDKN2B. Further analysis revealed that the inhibition of TGF-β1 signaling in inflamed epithelial cells by the small molecule could suppress the EMT-related markers as well as improve the expression of epithelial adherens and tight junctions. Collectively, these findings indicated that the inhibition of the TGF-β1 signaling could suppress the induction of EMT in inflamed epithelial cells as well as exert a protective effect on preserving tight junction integrity. There is a pressing need to determine the exact cellular mechanisms by which TGF-β1 exerts its effect on IBD pathogenesis.

SUBMITTER: Ghorbaninejad M

PROVIDER: S-EPMC10408554 | biostudies-literature | 2023 Apr

REPOSITORIES: biostudies-literature

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TGF-β receptor I inhibitor may restrict the induction of EMT in inflamed intestinal epithelial cells.

Ghorbaninejad Mahsa M Abdollahpour-Alitappeh Meghdad M Shahrokh Shabnam S Fayazzadeh Sara S Asadzadeh-Aghdaei Hamid H Meyfour Anna A

Experimental biology and medicine (Maywood, N.J.) 20230212 8

Despite the extensive body of research, understanding the exact molecular mechanisms governing inflammatory bowel diseases (IBDs) still demands further investigation. Transforming growth factor-β1 (TGF-β1) signaling possesses a multifacial effect on a broad range of context-dependent cellular responses. However, long-term TGF-β1 activity may trigger epithelial-mesenchymal transition (EMT), followed by fibrosis. This study aimed to determine the role of epithelial TGF-β1 signaling in inflammatory ...[more]

PMID: 36775873

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Project description:Fibroblast growth factor (FGF) and transforming growth factor-beta (TGF-β) can regulate and/or dysregulate lens epithelial cell (LEC) behaviour, including proliferation, fibre differentiation, and epithelial-mesenchymal transition (EMT). Earlier studies have investigated the crosstalk between FGF and TGF-β in dictating lens cell fate, that appears to be dose dependent. Here, we tested the hypothesis that a fibre-differentiating dose of FGF differentially regulates the behaviour of lens epithelial cells undergoing TGF-β-induced EMT. Postnatal 21-day-old rat lens epithelial explants were treated with a fibre-differentiating dose of FGF-2 (200 ng/mL) and/or TGF-β2 (50 pg/mL) over a 7-day culture period. We compared central LECs (CLECs) and peripheral LECs (PLECs) using immunolabelling for changes in markers for EMT (α-SMA), lens fibre differentiation (β-crystallin), epithelial cell adhesion (β-catenin), and the cytoskeleton (alpha-tropomyosin), as well as Smad2/3- and MAPK/ERK1/2-signalling. Lens epithelial explants cotreated with FGF-2 and TGF-β2 exhibited a differential response, with CLECs undergoing EMT while PLECs favoured more of a lens fibre differentiation response, compared to the TGF-β-only-treated explants where all cells in the explants underwent EMT. The CLECs cotreated with FGF and TGF-β immunolabelled for α-SMA, with minimal β-crystallin, whereas the PLECs demonstrated strong β-crystallin reactivity and little α-SMA. Interestingly, compared to the TGF-β-only-treated explants, α-SMA was significantly decreased in the CLECs cotreated with FGF/TGF-β. Smad-dependent and independent signalling was increased in the FGF-2/TGF-β2 co-treated CLECs, that had a heightened number of cells with nuclear localisation of Smad2/3 compared to the PLECs, that in contrast had more pronounced ERK1/2-signalling over Smad2/3 activation. The current study has confirmed that FGF-2 is influential in differentially regulating the behaviour of LECs during TGF-β-induced EMT, leading to a heterogenous cell population, typical of that observed in the development of post-surgical, posterior capsular opacification (PCO). This highlights the cooperative relationship between FGF and TGF-β leading to lens pathology, providing a different perspective when considering preventative measures for controlling PCO.

Project description:Under physiological conditions, CD4+ regulatory T (Treg) cells expressing the transcription factor Foxp3 are generated in the thymus [thymus-derived Foxp3+ Treg (tTregs) cells] and extrathymically at peripheral sites [peripherally induced Foxp3+ Treg (pTreg) cell], and both developmental subsets play non-redundant roles in maintaining self-tolerance throughout life. In addition, a variety of experimental in vitro and in vivo modalities can extrathymically elicit a Foxp3+ Treg cell phenotype in peripheral CD4+Foxp3- T cells, which has attracted much interest as an approach toward cell-based therapy in clinical settings of undesired immune responses. A particularly notable example is the in vitro induction of Foxp3 expression and Treg cell activity (iTreg cells) in initially naive CD4+Foxp3- T cells through T cell receptor (TCR) and IL-2R ligation, in the presence of exogenous TGF-β. Clinical application of Foxp3+ iTreg cells has been hampered by the fact that TGF-β-driven Foxp3 induction is not sufficient to fully recapitulate the epigenetic and transcriptional signature of in vivo induced Foxp3+ tTreg and pTreg cells, which includes the failure to imprint iTreg cells with stable Foxp3 expression. This hurdle can be potentially overcome by pharmacological interference with DNA methyltransferase activity and CpG methylation [e.g., by the cytosine nucleoside analog 5-aza-2'-deoxycytidine (5-aza-dC)] to stabilize TGF-β-induced Foxp3 expression and to promote a Foxp3+ iTreg cell phenotype even in the absence of added TGF-β. However, the molecular mechanisms of 5-aza-dC-mediated Foxp3+ iTreg cell generation have remained incompletely understood. Here, we show that in the absence of exogenously added TGF-β and IL-2, efficient 5-aza-dC-mediated Foxp3+ iTreg cell generation from TCR-stimulated CD4+Foxp3- T cells is critically dependent on TGF-βR and IL-2R signaling and that this process is driven by TGF-β and IL-2, which could either be FCS derived or produced by T cells on TCR stimulation. Overall, these findings contribute to our understanding of the molecular mechanisms underlying the process of Foxp3 induction and may provide a rational basis for generating phenotypically and functionally stable iTreg cells.

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TGF-β receptor I inhibitor may restrict the induction of EMT in inflamed intestinal epithelial cells.

Publications

TGF-β receptor I inhibitor may restrict the induction of EMT in inflamed intestinal epithelial cells.

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