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Quantitative Proteomic Analysis in Alveolar Type II Cells Reveals the Different Capacities of RAS and TGF-β to Induce Epithelial-Mesenchymal Transition.

ABSTRACT: Alveolar type II (ATII) epithelial cells function as stem cells, contributing to alveolar renewal, repair and cancer. Therefore, they are a highly relevant model for studying a number of lung diseases, including acute injury, fibrosis and cancer, in which signals transduced by RAS and transforming growth factor (TGF)-β play critical roles. To identify downstream molecular events following RAS and/or TGF-β activation, we performed proteomic analysis using a quantitative label-free approach (LC-HDMS^E) to provide in-depth proteome coverage and estimates of protein concentration in absolute amounts. Data are available via ProteomeXchange with identifier PXD023720. We chose ATII^ER:KRASV12 as an experimental cell line in which RAS is activated by adding 4-hydroxytamoxifen (4-OHT). Proteomic analysis of ATII cells treated with 4-OHT or TGF-β demonstrated that RAS activation induces an epithelial-mesenchymal transition (EMT) signature. In contrast, under the same conditions, activation of TGF-β signaling alone only induces a partial EMT. EMT is a dynamic and reversible biological process by which epithelial cells lose their cell polarity and down-regulate cadherin-mediated cell-cell adhesion to gain migratory properties, and is involved in embryonic development, wound healing, fibrosis and cancer metastasis. Thus, these results could help to focus research on the identification of processes that are potentially driving EMT-related human disease.

SUBMITTER: Zhou Y

PROVIDER: S-EPMC8048883 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Quantitative Proteomic Analysis in Alveolar Type II Cells Reveals the Different Capacities of RAS and TGF-β to Induce Epithelial–Mesenchymal Transition

Project description:Alveolar type II (ATII) epithelial cells function as stem cells, contributing to alveolar renewal, repair and cancer. Therefore, they are a highly relevant model for studying lung diseases, including acute injury, fibrosis and cancer, in which signals transduced by RAS and transforming growth factor (TGF)-ß play critical roles. To identify downstream molecular events following RAS and/or TGF-ß activation, we performed proteomic analysis using a quantitative label-free approach (LC-HDMSE) to provide in-depth proteome coverage and estimates of protein concentration in absolute amounts. Contact: pjss@soton.ac.uk

2021-02-04 | PXD023720 | Pride

CXCL9 Regulates TGF-?1-Induced Epithelial to Mesenchymal Transition in Human Alveolar Epithelial Cells.

Project description:Epithelial to mesenchymal cell transition (EMT), whereby fully differentiated epithelial cells transition to a mesenchymal phenotype, has been implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF). CXCR3 and its ligands are recognized to play a protective role in pulmonary fibrosis. In this study, we investigated the presence and extent of EMT and CXCR3 expression in human IPF surgical lung biopsies and assessed whether CXCR3 and its ligand CXCL9 modulate EMT in alveolar epithelial cells. Coexpression of the epithelial marker thyroid transcription factor-1 and the mesenchymal marker ?-smooth muscle actin and CXCR3 expression was examined by immunohistochemical staining of IPF surgical lung biopsies. Epithelial and mesenchymal marker expression was examined by quantitative real-time PCR, Western blotting, and immunofluorescence in human alveolar epithelial (A549) cells treated with TGF-?1 and CXCL9, with Smad2, Smad3, and Smad7 expression and cellular localization examined by Western blotting. We found that significantly more cells were undergoing EMT in fibrotic versus normal areas of lung in IPF surgical lung biopsy samples. CXCR3 was expressed by type II pneumocytes and fibroblasts in fibrotic areas in close proximity to cells undergoing EMT. In vitro, CXCL9 abrogated TGF-?1-induced EMT. A decrease in TGF-?1-induced phosphorylation of Smad2 and Smad3 occurred with CXCL9 treatment. This was associated with increased shuttling of Smad7 from the nucleus to the cytoplasm where it inhibits Smad phosphorylation. This suggests a role for EMT in the pathogenesis of IPF and provides a novel mechanism for the inhibitory effects of CXCL9 on TGF-?1-induced EMT.

| S-EPMC4777321 | biostudies-literature

IL-27 inhibits the TGF-β1-induced epithelial-mesenchymal transition in alveolar epithelial cells.

Project description:IL-27 is a multifunctional cytokine that has both pro-inflammatory and anti-inflammatory functions. Although IL-27 has been shown to potently inhibit lung fibrosis, the detailed mechanism of IL-27 in this process is poorly understood. Epithelial-mesenchymal transition (EMT) is one of the key mechanisms involved in pulmonary fibrosis. We assessed the effects of IL-27 on TGF-β1-induced EMT in alveolar epithelial cells.A549 cells (a human AEC cell line) were incubated with TGF-β1, IL-27, or both TGF-β1 and IL-27, and changes in E-cadherin, β-catenin, vimentin and a-SMA levels were measured using real-time PCR, western blotting and fluorescence microscopy. The related proteins in the JAK/STAT and TGF-β/Smad signalling pathways were examined by western blot.IL-27 increased the expression of epithelial phenotypic markers, including E-cadherin and β-catenin, and inhibited mesenchymal phenotypic markers, including vimentin and a-SMA in A549 cells. Moreover, TGF-β1-induced EMT was attenuated by IL-27. Furthermore, we found that TGF-β1 activated the phosphorylation of JAK1, STAT1, STAT3, STAT5, Smad1, Smad3 and Smad5, and IL-27 partially inhibited these changes in this process. When cells were treated with the STAT3 specific inhibitor wp1006 and the Smad3 specific inhibitor SIS3, the inhibition of EMT by IL-27 was significantly strengthened.Our results suggest that IL-27 attenuates epithelial-mesenchymal transition in alveolar epithelial cells in the absence or presence of TGF-β1 through the JAK/STAT and TGF-β/Smad signalling pathways.

| S-EPMC4774182 | biostudies-other

DA-Raf-Mediated Suppression of the Ras--ERK Pathway Is Essential for TGF-?1-Induced Epithelial-Mesenchymal Transition in Alveolar Epithelial Type 2 Cells.

Project description:Myofibroblasts play critical roles in the development of idiopathic pulmonary fibrosis by depositing components of extracellular matrix. One source of lung myofibroblasts is thought to be alveolar epithelial type 2 cells that undergo epithelial-mesenchymal transition (EMT). Rat RLE-6TN alveolar epithelial type 2 cells treated with transforming growth factor-?1 (TGF-?1) are converted into myofibroblasts through EMT. TGF-? induces both canonical Smad signaling and non-canonical signaling, including the Ras-induced ERK pathway (Raf-MEK-ERK). However, the signaling mechanisms regulating TGF-?1-induced EMT are not fully understood. Here, we show that the Ras-ERK pathway negatively regulates TGF-?1-induced EMT in RLE-6TN cells and that DA-Raf1 (DA-Raf), a splicing isoform of A-Raf and a dominant-negative antagonist of the Ras-ERK pathway, plays an essential role in EMT. Stimulation of the cells with fibroblast growth factor 2 (FGF2), which activated the ERK pathway, prominently suppressed TGF-?1-induced EMT. An inhibitor of MEK, but not an inhibitor of phosphatidylinositol 3-kinase, rescued the TGF-?1-treated cells from the suppression of EMT by FGF2. Overexpression of a constitutively active mutant of a component of the Ras-ERK pathway, i.e., H-Ras, B-Raf, or MEK1, interfered with EMT. Knockdown of DA-Raf expression with siRNAs facilitated the activity of MEK and ERK, which were only weakly and transiently activated by TGF-?1. Although DA-Raf knockdown abrogated TGF-?1-induced EMT, the abrogation of EMT was reversed by the addition of the MEK inhibitor. Furthermore, DA-Raf knockdown impaired the TGF-?1-induced nuclear translocation of Smad2, which mediates the transcription required for EMT. These results imply that intrinsic DA-Raf exerts essential functions for EMT by antagonizing the TGF-?1-induced Ras-ERK pathway in RLE-6TN cells.

| S-EPMC4440819 | biostudies-literature

Regulatory T Cells Induce Metastasis by Increasing Tgf-? and Enhancing the Epithelial–Mesenchymal Transition.

Project description:Malignant melanoma is the most aggressive form of skin cancer; a substantial percentage of patients present with distant metastases. However, the mechanism of metastasis is not well understood. Here, we demonstrate that the administration of exogenous regulatory T cells (Tregs) into melanoma tumor-bearing mice results in a significant increase in lung metastasis. An increase in the invasive and metastatic phenotype of melanoma was mediated by cell-to-cell contact between melanoma cells and Tregs, which elevated the expression level of transforming growth factor-? (TGF-?) and the subsequent induction of the epithelial-to-mesenchymal transition (EMT).?B16-BL6 melanoma tumors co-cultured with Tregs showed a larger population of migrating cells compared to B16-BL6 tumors cultured without Tregs. Additionally, the injection of exogenous Tregs into B16-BL6 melanoma tumors led to the recruitment and infiltration of endogenous Tregs into tumor tissues, thus increasing the overall Treg percentage in the tumor infiltrating lymphocyte population. Collectively, our findings propose novel mechanisms in which exogenous Treg-dependent upregulation of TGF-? and mesenchymal markers is important for augmenting the migration capacity and invasiveness of melanoma, thereby contributing to the metastasis.

| S-EPMC6912455 | biostudies-literature

Quantitative peptidomics of endogenous peptides involved in TGF-?1-induced epithelial mesenchymal transition of renal epithelial cells.

Project description:TGF-?1 is a key fibrotic factor mediating epithelial mesenchymal transition (EMT) of epithelial cells through various signaling pathways. However, roles of proteolytic cleavage and endogenous peptide dynamics in TGF-?1-induced EMT remain unknown. We therefore performed quantitative peptidomics of TGF-?1-induced EMT in renal tubular epithelial cells. The acquired mesenchymal characteristics were confirmed, including morphological change (from cobblestone-like to fibroblast-like), decreased epithelial marker (ZO-1), and increased mesenchymal marker (vimentin). Quantitative peptidomics using stable isotope labeling revealed significantly altered levels of 70 unique endogenous peptides (derived from internal and C-terminal parts of 39 unique precursor proteins) after EMT induction. Interestingly, the majority of these peptides were derived from non-short-lived proteins, and analysis of P1 position revealed predominance of hydrophobic residues, suggesting that these endogenous peptides were generated mainly from proteasome cleavage. This hypothesis was confirmed by treating the cells with MG132 (a proteasome inhibitor), which provided almost identical endogenous peptide pattern as of the TGF-?1-treated cells. Moreover, validation assay showed marked reduction of proteasome peptidase activity in both TGF-?1-treated and MG132-treated cells. This is the first peptidome dataset that provides several novel aspects of mechanisms for TGF-?1-induced EMT. Our data also suggest that TGF-?1 exerts inhibitory effect against proteasome activity during EMT induction.

| S-EPMC5841401 | biostudies-literature

Atractylodin Suppresses TGF-β-Mediated Epithelial-Mesenchymal Transition in Alveolar Epithelial Cells and Attenuates Bleomycin-Induced Pulmonary Fibrosis in Mice.

Project description:Idiopathic pulmonary fibrosis (IPF) is characterized by fibrotic change in alveolar epithelial cells and leads to the irreversible deterioration of pulmonary function. Transforming growth factor-beta 1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) in type 2 lung epithelial cells contributes to excessive collagen deposition and plays an important role in IPF. Atractylodin (ATL) is a kind of herbal medicine that has been proven to protect intestinal inflammation and attenuate acute lung injury. Our study aimed to determine whether EMT played a crucial role in the pathogenesis of pulmonary fibrosis and whether EMT can be utilized as a therapeutic target by ATL treatment to mitigate IPF. To address this topic, we took two steps to investigate: 1. Utilization of anin vitro EMT model by treating alveolar epithelial cells (A549 cells) with TGF-β1 followed by ATL treatment for elucidating the underlying pathways, including Smad2/3 hyperphosphorylation, mitogen-activated protein kinase (MAPK) pathway overexpression, Snail and Slug upregulation, and loss of E-cadherin. Utilization of an in vivo lung injury model by treating bleomycin on mice followed by ATL treatment to demonstrate the therapeutic effectiveness, such as, less collagen deposition and lower E-cadherin expression. In conclusion, ATL attenuates TGF-β1-induced EMT in A549 cells and bleomycin-induced pulmonary fibrosis in mice.

| S-EPMC8570326 | biostudies-literature

Cancer-associated fibroblasts induce epithelial-mesenchymal transition of breast cancer cells through paracrine TGF-β signalling.

Project description:BackgroundCancer-associated fibroblasts (CAFs) activated by tumour cells are the predominant type of stromal cells in breast cancer tissue. The reciprocal effect of CAFs on breast cancer cells and the underlying molecular mechanisms are not fully characterised.MethodsStromal fibroblasts were isolated from invasive breast cancer tissues and the conditioned medium of cultured CAFs (CAF-CM) was collected to culture the breast cancer cell lines MCF-7, T47D and MDA-MB-231. Neutralising antibody and small-molecule inhibitor were used to block the transforming growth factor-β (TGF-β) signalling derived from CAF-CM, which effect on breast cancer cells.ResultsThe stromal fibroblasts isolated from breast cancer tissues showed CAF characteristics with high expression levels of α-smooth muscle actin and SDF1/CXCL12. The CAF-CM transformed breast cancer cell lines into more aggressive phenotypes, including enhanced cell-extracellular matrix adhesion, migration and invasion, and promoted epithelial-mesenchymal transition (EMT). Cancer-associated fibroblasts secreted more TGF-β1 than TGF-β2 and TGF-β3, and activated the TGF-β/Smad signalling pathway in breast cancer cells. The EMT phenotype of breast cancer cells induced by CAF-CM was reversed by blocking TGF-β1 signalling.ConclusionCancer-associated fibroblasts promoted aggressive phenotypes of breast cancer cells through EMT induced by paracrine TGF-β1. This might be a common mechanism for acquiring metastatic potential in breast cancer cells with different biological characteristics.

| S-EPMC3915130 | biostudies-literature

Mesenchymal stromal cells induce epithelial-to-mesenchymal transition in human colorectal cancer cells through the expression of surface-bound TGF-?.

Project description:Mesenchymal stem/stromal cells (MSC) are multipotent precursors endowed with the ability to home to primary and metastatic tumor sites, where they can integrate into the tumor-associated stroma. However, molecular mechanisms and outcome of their interaction with cancer cells have not been fully clarified. In this study, we investigated the effects mediated by bone marrow-derived MSC on human colorectal cancer (CRC) cells in vitro and in vivo. We found that MSC triggered epithelial-to-mesenchymal transition (EMT) in tumor cells in vitro, as indicated by upregulation of EMT-related genes, downregulation of E-cadherin and acquisition of mesenchymal morphology. These effects required cell-to-cell contact and were mediated by surface-bound TGF-? newly expressed on MSC upon coculture with tumor cells. In vivo tumor masses formed by MSC-conditioned CRC cells were larger and characterized by higher vessel density, decreased E-cadherin expression and increased expression of mesenchymal markers. Furthermore, MSC-conditioned tumor cells displayed increased invasiveness in vitro and enhanced capacity to invade peripheral tissues in vivo. Thus, by promoting EMT-related phenomena, MSC appear to favor the acquisition of an aggressive phenotype by CRC cells.

| S-EPMC4338537 | biostudies-literature

Fibronectin fibrils regulate TGF-?1-induced Epithelial-Mesenchymal Transition.

Project description:Epithelial-Mesenchymal Transition (EMT) is a dynamic process through which epithelial cells transdifferentiate from an epithelial phenotype into a mesenchymal phenotype. Previous studies have demonstrated that both mechanical signaling and soluble growth factor signaling facilitate this process. One possible point of integration for mechanical and growth factor signaling is the extracellular matrix. Here we investigate the role of the extracellular matrix (ECM) protein fibronectin (FN) in this process. We demonstrate that inhibition of FN fibrillogenesis blocks activation of the Transforming Growth Factor-Beta (TGF-?) signaling pathway via Smad2 signaling, decreases cell migration and ultimately leads to inhibition of EMT. Results show that soluble FN, FN fibrils, or increased contractile forces are insufficient to independently induce EMT. We further demonstrate that inhibition of latent TGF-?1 binding to FN fibrils via either a monoclonal blocking antibody against the growth factor binding domain of FN or through use of a FN deletion mutant that lacks the growth factor binding domains of FN blocks EMT progression, indicating a novel role for FN in EMT in which the assembly of FN fibrils serves to localize TGF-?1 signaling to drive EMT.

| S-EPMC5438896 | biostudies-literature

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