Epithelial-mesenchymal Transition In Alveolar Epithelial Cells Research Articles

Circular RNA MKLN1 promotes epithelial-mesenchymal transition in pulmonary fibrosis by regulating the miR-26a/b-5p/CDK8 axis in human alveolar epithelial cells and mice models.

Pulmonary fibrosis involves destruction of the lung parenchyma and extracellular matrix deposition. Effective treatments for pulmonary fibrosis are lacking and its pathogenesis is still unclear. Studies have found that epithelial-mesenchymal transition (EMT) of alveolar epithelial cells (AECs) plays an important role in progression of pulmonary fibrosis. Thus, an in-depth exploration of its mechanism might identify new therapeutic targets. In this study, we revealed that a novel circular RNA, MKLN1 (circMKLN1), was significantly elevated in two pulmonary fibrosis models (intraperitoneally with PQ, 50mg/kg for 7days, and intratracheally with BLM, 5mg/kg for 28days). Additionally, circMKLN1 was positively correlated with the severity of pulmonary fibrosis. Inhibition of circMKLN1 expression significantly reduced collagen deposition and inhibited EMT in AECs. EMT was aggravated after circMKLN1 overexpression in AECs. MiR-26a-5p/miR-26b-5p (miR-26a/b), the targets of circMKLN1, were confirmed by luciferase reporter assays. CircMKLN1 inhibition elevated miR-26a/b expression. Significantly decreased expression of CDK8 (one of the miR-26a/b targets) was observed after inhibition of circMKLN1. EMT was exacerbated again, and CDK8 expression was significantly increased after circMKLN1 inhibition and cotransfection of miR-26a/b inhibitors in AECs. Our research indicated that circMKLN1 promoted CDK8 expression through sponge adsorption of miR-26a/b, which regulates EMT and pulmonary fibrosis. This study provides a theoretical basis for finding new targets or biomarkers in pulmonary fibrosis.

Increased expression of OPN contributes to idiopathic pulmonary fibrosis and indicates a poor prognosis

BackgroundIdiopathic pulmonary fibrosis (IPF) is fibrotic lung disease with no effective treatment. It is characterized by destruction of alveolar structure and pulmonary interstitial fibrosis, leading to dyspnea and even asphyxia death of patients. Epithelial-mesenchymal transition (EMT) is considered to be a driving factor in the pathogenesis of IPF. Osteopontin (OPN) is a secreted protein widely present in the extracellular matrix and involved in the occurrence and development of a variety of diseases.MethodsThe original datasets were obtained from NCBI GEO databases analyzed with the online tool GEO2R and EasyGEO. Bleomycin induced mouse pulmonary fibrosis model and OPN/OPN-biotin treated mouse model were established to investigate the role of OPN in mouse pulmonary fibrosis and the target cells of OPN. A549 cells and HBE cells were used to explore the mechanism of OPN-induced epithelial-mesenchymal transition (EMT) in epithelial cells and mass spectrometry was used to detect OPN downstream receptors. Precision-cut lung slices and lentivirus-treated mice with pulmonary fibrosis were used to examine the therapeutic effect of OPN and its downstream pathways on pulmonary fibrosis.ResultsWe demonstrate that the content of OPN in IPF bronchoalveolar lavage fluid (BALF) is high compared to the normal groups, and its expression level is correlated with prognosis. At the animal level, OPN was highly expressed at all stages of pulmonary fibrosis in mice, and the bronchoalveolar lavage fluid (BALF) could accurately reflect its expression in the lung. Next, we reveal that OPN was mainly expressed by macrophages and the main target cells of OPN were epithelial cells. Mice developed pulmonary fibrosis accompanied after treating the mice with OPN. Both in vitro and in vivo experiments confirmed that OPN could induce EMT of alveolar epithelial cells. Mechanistically, OPN binding triggered phosphorylation of FAK by CD44, thus activating snail1-mediated profibrotic protein synthesis. Inhibition of FAK phosphorylation and its downstream pathways can effectively alleviate pulmonary fibrosis in precision sections of lung tissue (PCLS) assay. OPN knockdown in bleomycin-induced lung fibrosis mice led to significantly less fibrosis.ConclusionOur data suggest that OPN mediates lung fibrosis through EMT, implicating its potential therapeutic target and prognostic indicator role for IPF. OPN may be a target for the diagnosis and treatment of IPF.

PD-L1 upregulation promotes drug-induced pulmonary fibrosis by inhibiting vimentin degradation

Idiopathic pulmonary fibrosis (IPF) is a progressive disease with high mortality and limited therapeutic options. The immune checkpoint PD1/PD-L1 axis is related to the pathogenesis of pulmonary fibrosis, and upregulated expression levels of PD-L1 have been demonstrated in IPF patients. However, the mechanism of PD-L1 in pulmonary fibrosis is not fully understood. Here, we demonstrated upregulated expression of PD-L1 in fibrotic lung tissues and sera of IPF patients. Bleomycin (BLM) treatment induced PD-L1 upregulation, EMT (Epithelial-Mesenchymal Transition) and fibrosis-like morphology changes in human pulmonary alveolar epithelial cells (HPAEpiCs). Silencing PD-L1 attenuated BLM-induced EMT and fibrosis-like morphology changes in HPAEpiCs. In addition, we identified that PD-L1 directly binds to vimentin and inhibits vimentin ubiquitination, thereby increasing vimentin levels in HPAEpiCs. Silencing of vimentin inhibited BLM- and PD-L1-induced fibrosis in HPAEpiCs. The correlation between PD-L1 and EMT or vimentin expression was further confirmed in clinical samples and animal models. Finally, we used BLM- and paraquat-induced pulmonary fibrosis animal models to confirm the anti-pulmonary fibrosis effects of PD-L1 silencing. Taken together, our findings suggest that upregulated PD-L1 stimulates EMT of alveolar epithelial cells by increasing vimentin levels by inhibiting vimentin ubiquitination, thereby contributing to pulmonary fibrosis.

MALAT1-miR-30c-5p-CTGF/ATG5 axis regulates silica-induced experimental silicosis by mediating EMT in alveolar epithelial cells

Epithelial-mesenchymal transdifferentiation of alveolar type Ⅱ epithelial cells is a vital source of pulmonary myofibroblasts, and myofibroblasts formation is recognized as an important phase in the pathological process of silicosis. miR-30c-5p has been determined to be relevant in the activation of the epithelial-mesenchymal transition (EMT) in numerous disease processes. However, elucidating the role played by miR-30c-5p in the silicosis-associated EMT process remains a great challenge. In this work, based on the establishment of mouse silicosis and A549 cells EMT models, miR-30c-5p was interfered with in vivo and in vitro models to reveal its effects on EMT and autophagy. Moreover, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), connective tissue growth factor (CTGF), autophagy-related gene 5 (ATG5), and autophagy were further interfered with in the A549 cells models to uncover the possible molecular mechanism through which miR-30c-5p inhibits silicosis associated EMT. The results demonstrated the targeted binding of miR-30c-5p to CTGF, ATG5, and MALAT1, and showed that miR-30c-5p could prevent EMT in lung epithelial cells by acting on CTGF and ATG5-associated autophagy, thereby inhibiting the silicosis fibrosis process. Furthermore, we also found that lncRNA MALAT1 might competitively absorb miR-30c-5p and affect the EMT of lung epithelial cells. In a word, interfering with miR-30c-5p and its related molecules (MALAT1, CTGF, and ATG5-associated autophagy) may provide a reference point for the application of silicosis intervention-related targets.

Effect of Baricitinib on the epithelial-mesenchymal transition of alveolar epithelial cells induced by IL-6

ObjectiveInterstitial lung disease (ILD) is one of the common complications of Connective tissue disease (CTD). Epithelial-mesenchymal transition (EMT) is one of the main pathological mechanisms of ILD. IL-6 may induce ILD through the JAK/STAT pathway. Therefore, exploring the mechanism of IL-6 on the EMT of alveolar epithelial cells and inhibition JAK/STAT pathway with Baricitinib on the EMT of alveolar epithelial cells is helpful in revealing the pathogenesis of CTD-ILD and guiding treatment. MethodsElectrochemiluminescence was applied to detect the changes in serum IL-6 levels before and after treatment in 37 patients with anti-synthetase syndrome-associated ILD; A549 cells (a human AEC cell line) were incubated with IL-6, Baricitinib, or both IL-6 and Baricitinib, and changes in EMT-related markers levels were measured using real-time PCR, western blotting and fluorescence microscopy. The related proteins in the JAK/STAT signaling pathways were examined by western blot. The level of Connective tissue growth factor (CTGF) and Hydroxyproline (Hyp) in cell supernatants was measured by ELISA. ResultsSerum IL-6 level in patients with anti-synthetase syndrome-associated ILD was significantly higher than that in health (6.78(4.19, 16.14)pg/ml vs. 2.10(1.43, 5.18)pg/ml, p < 0.01). The level of IL-6 in the improvement group of ASS-ILD was considerably decreased than that before treatment(before(7.48(4.54, 22.76) pg/mL vs. 5.00(3.46, 11.32)pg/mL, p < 0.01), p < 0.01), and the level of IL-6 in the progressive group of ASS-ILD was significantly higher than that before treatment(before(7.49(6.77, 35.80) pg/mL vs. 30.02(8.01, 82.98) pg/mL, p < 0.05). IL-6 increased the expression of epithelial phenotypic marker E-cadherin and inhibited mesenchymal phenotypic markers, including vimentin and N-cadherin in A549 cells. Moreover, IL-6-induced EMT was attenuated by Baricitinib. Furthermore, we found that IL-6 activated the phosphorylation of JAK1/2, STAT3, and Baricitinib, partially inhibiting these changes in this process. Baricitinib reduced the secretion of CTGF and Hyp in A549 cells. ConclusionThe significant higher level of IL-6 in patients with anti-synthase syndrome-associated ILD may be related to disease activity and recurrence. Our results suggest that Baricitinib attenuates epithelial-mesenchymal transition in alveolar epithelial cells in the presence of IL-6 through the JAK/STAT signaling pathway.

Role of integrin α2 in methotrexate-induced epithelial-mesenchymal transition in alveolar epithelial A549 cells.

Methotrexate (MTX) is widely used to treat various diseases. However, it induces adverse reactions like serious lung injury, including pulmonary fibrosis. Increasing evidence suggests that epithelial-mesenchymal transition (EMT) in injured alveolar epithelium contributes to the development of the pathophysiological state of the lung. We demonstrated that MTX induced EMT in cultured alveolar epithelial cell lines. Integrin-mediated signaling is considered a significant factor in recognizing the EMT process. However, the relationship between MTX-induced EMT and integrin family members is poorly understood. In the present study, we aimed to clarify the role of integrin in MTX-induced EMT in A549 and NCI-H1299 (H1299) cells by focusing on the integrin alpha 2 (ITGA2) subunit, selected based on our microarray analysis. MTX treatment for 72h significantly increased the mRNA and cell surface expression of ITGA2 in both cell lines. However, this upregulation by MTX was suppressed by co-treatment with SB431542 and folic acid, which are inhibitors of MTX-induced EMT in A549 cells. The mRNA expression levels of EMT-related genes were more affected in the MTX-treated A549 cells with high ITGA2 expression than in those with low ITGA2 expression. Finally, E7820, an ITGA2 inhibitor, suppressed MTX-induced EMT-related phenotypic changes, such as morphology and mRNA and protein expression of α-smooth muscle actin, a representative EMT marker. These findings suggest that ITGA2 may play a key role in MTX-induced EMT in alveolar epithelial cells.

Delicate regulation of IL-1β-mediated inflammation by cyclophilin A.

The inflammatory response is tightly regulated, but its regulatory principles are still incompletely understood. Cyclophilin A (CypA) has long been considered as a pro-inflammatory factor. Here, we discover how CypA precisely regulates interleukin-1β (IL-1β)-mediated inflammatory responses. In lipopolysaccharide-treated mice, CypA deficiency initially inhibits and then promotes lung inflammation, which is closely related to IL-1β production. Mechanistically, CypA not only facilitates pro-IL-1β processing by increasing Smurf1-mediated K63-linked ubiquitination in an ATP-dependent manner but also accelerates pro-IL-1β degradation, depending on Smurf1-mediated K48-linked ubiquitination. Moreover, in IL-1β-treated mice, CypA exacerbates lung injury by enhancing cytokine production. It also upregulates the ILK/AKT pathway by inhibiting Cyld-mediated K63-linked ILK deubiquitination, which promotes the epithelial-mesenchymal transition (EMT) to facilitate lung repair. Collectively, CypA promotes inflammation activation by increasing IL-1β production and then promotes inflammation resolution by enhancing redundant pro-IL-1β degradation and IL-1β-induced EMT, indicating the complex and delicate regulation of inflammatory response.

Vasoactive intestinal peptide attenuates bleomycin-induced murine pulmonary fibrosis by inhibiting epithelial-mesenchymal transition: Restoring autophagy in alveolar epithelial cells

Vasoactive intestinal peptide (VIP) is an intrapulmonary neuropeptide with multi-function, including anti-fibrosis. However, the exact role of VIP in pulmonary fibrosis has not been documented. Here, we investigated the protective effect of VIP against pulmonary fibrosis in a murine model induced by bleomycin (BLM). We found that the overexpression of VIP mediated by the adenoviral vector significantly attenuated the lung tissue destruction, reduced the deposition of the extracellular matrix, and inhibited the expression of alpha-smooth muscle actin (α-SMA) in the lungs of mice received BLM. Mechanismly, we found that VIP significantly suppressed the transforming growth factor-beta 1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) and inhibited the matrix-producing ability of alveolar epithelial cells in vitro. Furthermore, we found that TGF-β1 depressed the autophagy and an autophagy inductor partly reversed the TGF-β1-induced EMT in alveolar epithelial cells. The impaired autophagy was also observed in the lungs of BLM-treated mice, which was restored by VIP treatment. And VIP treatment enhanced autophagy in TGF-β1-stimulated alveolar epithelial cells, contributing to its anti-EMT effect. In summary, our data, for the first time, show that VIP attenuates BLM-induced pulmonary fibrosis in mice with anti-EMT effect through restoring autophagy in alveolar epithelial cells. This study provides a possibility that inhaled long-acting VIP may be an anti-fibrotic drug in the treatment of pulmonary fibrosis.

IL-23 amplifies the epithelial-mesenchymal transition of mechanically conditioned alveolar epithelial cells in rheumatoid arthritis-associated interstitial lung disease through mTOR/S6 signaling.

Epithelial-mesenchymal transition (EMT) creates an environment facilitating fibrosis following alveolar epithelial cell injury. IL-23 has important roles in chronic autoimmune conditions like rheumatoid arthritis (RA), but its role in the interstitial lung disease that affects patients with RA is unclear. This study aimed to determine the profibrogenic role of IL-23 on somatic alveolar type I (ATI) epithelial cells. Primary ATI cells were isolated from rats and cultured on plastic dishes for 1-3 wk. After prolonged culture (≥14 days) on rigid culture dishes, primary ATI cells gradually acquired a mesenchymal phenotype, identified by decreased expression of caveolin-1, and reorganization of F-actin cytoskeleton, indicating the initiation of EMT by matrix stiffness. To determine how IL-23 promotes EMT in vitro, transitioning ATI cells, cultured on a stiff substrate for ≥14 days were stimulated with IL-23. The EMT phenotype was significantly enhanced by IL-23, which upregulated α-smooth muscle actin (α-SMA), collagen I/III protein, and decreased caveolin-1. Furthermore, IL-23 significantly promoted cell invasion, as well as apoptotic resistance on transitioning ATI cells. Mechanistically, IL-23-induced EMT was mammalian target of rapamycin/ribosomal protein S6 (mTOR/S6) signaling dependent and reversible by rapamycin. Transcriptional sequencing analysis of human lung fibrosis biopsy tissue revealed key roles for IL-23 in rheumatoid arthritis-associated interstitial lung disease (RA-ILD). This result was further validated by significantly upregulated IL-23 expression at the mRNA level in RA-ILD lung sections. Notably, transitioning ATI epithelial cells were abundantly detected in RA-ILD tissue. Taken together, these data support a role for IL-23 in the pathogenesis of RA lung fibrosis by promoting EMT in alveolar epithelial cells through mTOR/S6 signaling.

Evaluation on epithelial-mesenchymal state and microRNAs focusing on isolated alveolar epithelial cells from bleomycin injured rat lung

Several studies using bleomycin (BLM)-induced lung injury rat model revealed that epithelial-mesenchymal transition (EMT) contributes to pulmonary fibrosis. Conversely, microRNAs (miRNAs) are considered as useful markers of various diseases. In the present study, we aimed to characterize the EMT state through focusing on alveolar epithelial cells and identify the miRNAs that can be used as markers to predict pulmonary fibrosis using a BLM-induced lung injury rat model. Intratracheal administration of BLM increased hydroxyproline, a component of collagen, in lung tissues at day 14, but not at day 7. However, BLM induced EMT at day 7, which was accompanied with increased mRNA expression of α-smooth muscle actin, a representative EMT marker, in alveolar epithelium, thereby suggesting that EMT occurs prior to pulmonary fibrosis in alveolar epithelial cells. Using this rat model, the expression levels of several EMT-associated miRNAs were examined, and miR-222 was found to be upregulated in alveolar epithelial cells as well as bronchoalveolar lavage fluid from day 3. Our findings indicate that EMT in alveolar epithelial cells may occur before pulmonary fibrosis, and miR-222 may be used as a potential marker for early prediction of pulmonary fibrosis.

Interleukin-27 Inhibits Epithelial-Mesenchymal Transition in Ovalbumin-Induced Mice Bronchial Epithelial Cells

The epithelial-mesenchymal transition (EMT) of bronchial epithelial cells is a critical mechanism involved in transforming growth factor beta 1 (TGF-β1) induced asthma airway remodeling. Previous study has shown that interleukin 27 (IL-27) attenuates EMT in alveolar epithelial cells, but its effects on the BEAS-2B human bronchial epithelial cell line EMT remain unknown. Herein, we explored the effects of IL-27 on BEAS-2B EMT in vivo and in vitro. In the in vivo experiments, we found that IL-27 nose-drip therapy alleviated airway remodeling, increased the epithelial phenotypic marker epithelial-cadherin (E-cadherin), and decreased the mesenchymal phenotypic marker alpha-smooth muscle actin (α-SMA) compared with the asthmatic control group. We also found that IL-27 suppressed the signal transducer and activator of transcription (STAT3) in the lung tissue of asthmatic mice. in vitro, TGF-β1-induced EMT changes, including downregulation of E-cadherin and upregulation of α-SMA, were suppressed by IL-27 treatment. Additionally, STAT3 phosphorylation was activated by TGF-β1, whereas IL-27 inhibited the activation of TGF-β1 induced STAT3 phosphorylation. Our findings indicated that IL-27 could inhibit airway remodeling by attenuating bronchial epithelial cell EMT in vivo and in vitro. Therefore, IL-27 may be a beneficial therapeutic option targeting asthmatic airway remodeling.

LncRNA CHRF promotes TGF-β1 induced EMT in alveolar epithelial cells by inhibiting miR-146a up-regulating L1CAM expression

Purpose Idiopathic pulmonary fibrosis (IPF) is a type of progressive lung fibrosis disease. The survival time of diagnosed IPF patients is often only 2 years. Currently much evidence showed that the epithelial–mesenchymal transition (EMT) process is the main cause of the occurrence and development of IPF. LncRNA cardiac hypertrophy related factor (CHRF) was reported to be related with IPF development. Here we explored the functions and regulatory mechanisms of CHRF on EMT in IPF. Materials and methods A549 cells were treated with transforming growth factor-β1 (TGF-β1) for 48 h to construct IPF cell model. CHRF and miR-146a expression were quantified using qPCR. The expression of L1 cell adhesion molecule (L1CAM) and EMT related indicators (E-cadherin, Vimentin, Slug and N-cadherin) were detected by qPCR and western blot. Dual luciferase reporter experiment was conducted to prove the molecular interaction of miR-146a and L1CAM, as well as CHRF and miR-146a. Results CHRF and L1CAM expression were significantly upregulated and promoted the EMT process in A549 after treatment of TGF-β1. MiR-146a was obviously down-regulated, and knockdown of CHRF inhibited the EMT process by up-regulating miR-146a, in A549 after treatment of TGF-β1. Meanwhile, overexpression of miR-146a inhibited EMT process via targeting L1CAM. In addition, L1CAM overexpression eliminated the inhibitory effect of sh-CHRF on the EMT process. Conclusions These results provided evidence that CHRF promoted EMT process in A549 after treatment of TGF-β1, which proposed a new insight for depth understanding the pathological mechanisms of IPF.

Chrysin inhibited epithelial-mesenchymal transition of type Ⅱ alveolar epithelial cell by regulating NF-κB/Twist 1 signaling pathway

This study aimed to assess whether chrysin(ChR) can inhibit epithelial-mesenchymal transition(EMT) of type Ⅱ alveolar epithelial cell and produce anti-pulmonary fibrosis effect by regulating the NF-κB/Twist 1 signaling pathway. Sixty rats were randomly divided into the control group, the bleomycin(BLC) group, BLC+ChR(50 mg·kg~(-1)) group and BLC+ChR(100 mg·kg~(-1)) group, with 15 rats in each group. The pulmonary fibrosis model was induced by intratracheal injection of BLC(7 500 U·kg~(-1)). Rats were orally administered with different doses of ChR after BLC injection for 28 days. The cells were divided into control group, TGF-β1 group(5 ng·mL~(-1)), and TGF-β1+ChR(1, 10, 100 μmol·L~(-1)) groups. The type Ⅱ alveolar epithelial cells were treated with TGF-β1 for 24 h, and then treated with TGF-β1 for 48 h in the presence or absence of different doses of ChR(1, 10 and 100 μmol·L~(-1)). The morphological changes and collagen deposition in lung tissues were analyzed by HE staining, Masson staining and immunohistochemistry. The mRNA and protein expression levels of collagen Ⅰ, E-cadherin, zonula occludens-1(ZO-1), vimentin, alpha smooth muscle actin(α-SMA), inhibitor of nuclear factor kappa B alpha(IκBα), nuclear factor-kappa B p65(NF-κB p65), phospho-NF-κB p65(p-p65) and Twist 1 in lung tissues and cells were detected by qPCR and Western blot, respectively. The animal experiment results showed that as compared with the BLC group, after administration of ChR for 28 days, bleomycin-induced pulmonary fibrosis in rats was significantly relieved, collagen Ⅰ expression in lung tissues was significantly reduced(P&lt;0.05 or P&lt;0.01), and EMT of alveolar epithelial cells was obviously inhibited [the expression levels of E-cadherin and ZO-1 were increased and the expression levels of vimentin and α-SMA were decreased(P&lt;0.05 or P&lt;0.01)], concomitantly with significantly reduced IκBα and p65 phosphorylation level in cytoplasm and decreased NF-κB p65 and Twist 1 expression in nucleus(P&lt;0.05 or P&lt;0.01). The cell experiment results showed that different doses of ChR(1, 10 and 100 μmol·L~(-1)) significantly reduced TGF-β1-induced collagen Ⅰ expression(P&lt;0.05 or P&lt;0.01), significantly inhibited EMT of type Ⅱ alveolar epithelial cells[the expression levels of E-cadherin and ZO-1 were increased and the expression levels of vimentin and α-SMA were decreased(P&lt;0.05 or P&lt;0.01)], and inhibited IκBα and p65 phosphorylation in cytoplasm and down-regulated NF-κB p65 and Twist 1 expression in nucleus induced by TGF-β1(P&lt;0.05 or P&lt;0.01). The results suggest that ChR can reverse EMT of type Ⅱ alveolar epithelial cell and alleviate pulmonary fibrosis in rats, and its mechanism may be associated with reducing IκBα phosphorylation and inhibiting NF-κB p65 phosphorylation and nuclear transfer, thus down-regulating Twist 1 expression.

Andrographolide attenuates epithelial‐mesenchymal transition induced by TGF‐β1 in alveolar epithelial cells

Andrographolide (Andro), a component from Chinese medicinal herb Andrographis paniculata, could alleviate pulmonary fibrosis in rodents. Yet, whether and how Andro mitigates epithelial‐mesenchymal transition (EMT) induced by TGF‐β1 remain unknown. This study aimed to explore the effect of Andro on TGF‐β1‐induced EMT in human alveolar epithelial cells (AECs) and the mechanisms involved. We illustrated that Andro inhibited TGF‐β1‐induced EMT and EMT‐related transcription factors in alveolar epithelial A549 cells. Andro also reduced TGF‐β1‐induced cell migration and synthesis of pro‐fibrotic factors (ie CCN‐2, TGF‐β1), matrix metalloproteinases (ie MMP‐2, MMP‐9) and extracellular matrix (ECM) components (ie collagen 1), implying the inhibiting effect of Andro on TGF‐β1‐induced EMT‐like cell behaviours. Mechanistically, Andro treatment not only repressed TGF‐β1‐induced Smad2/3 phosphorylation and Smad4 nuclear translocation, but also suppressed TGF‐β1‐induced Erk1/2 phosphorylation and nuclear translocation in A549 cells. And treatment with ALK5 inhibitor (SB431542) or Erk1/2 inhibitors (SCH772984 and PD98059) remarkably reduced EMT evoked by TGF‐β1. In addition, Andro also reduced TGF‐β1‐induced intracellular ROS generation and NOX4 expression, and elevated antioxidant superoxide dismutase 2 (SOD2) expression, demonstrating the inhibiting effect of Andro on TGF‐β1‐induced oxidative stress, which is closely linked to EMT. Furthermore, Andro remarkably attenuated TGF‐β1‐induced down‐regulation of sirtuin1 (Sirt1) and forkhead box O3 (FOXO3), implying that Andro protects AECs from EMT partially by activating Sirt1/FOXO3‐mediated anti‐oxidative stress pathway. In conclusion, Andro represses TGF‐β1‐induced EMT in AECs by suppressing Smad2/3 and Erk1/2 signalling pathways and is also closely linked to the activation of sirt1/FOXO3‐mediated anti‐oxidative stress pathway.

1α, 25-dihydroxyvitamin D3 inhibits transforming growth factor β1-induced epithelial-mesenchymal transition via β-catenin pathway.

Background:The transforming growth factor β1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) has been proven associated with the pathogenesis of asthmatic airway remodeling, in which the Wnt/β-catenin pathway plays an important role, notably with regard to TGF-β1. Recent studies have shown that 1α, 25-dihydroxyvitamin D3(1α, 25(OH)2D3) inhibits TGF-β1-induced EMT, although the underlying mechanism have not yet been fully elucidated.Methods:Alveolar epithelial cells were exposed to 1α, 25(OH)2D3, ICG-001, or a combination of both, followed by stimulation with TGF-β1. The protein expression of E-cadherin, α-smooth muscle actin, fibronectin, and β-catenin was analyzed by western blotting and immunofluorescence analysis. The mRNA transcript of Snail was analyzed using RT-qPCR, and matrix metalloproteinase 9 (MMP-9) activity was analyzed by gelatin zymogram. The activity of the Wnt/β-catenin signaling pathway was analyzed using the Top/Fop flash reporters.Results:Both 1α, 25(OH)2D3 and ICG-001 blocked TGF-β1-induced EMT in alveolar epithelial cells. In addition, the Top/Fop Flash reporters showed that 1α, 25(OH)2D3 suppressed the activity of the Wnt/β-catenin pathway and reduced the expression of target genes, including MMP-9 and Snail, in synergy with ICG-001.Conclusion:1α, 25(OH)2D3 synergizes with ICG-001 and inhibits TGF-β1-induced EMT in alveolar epithelial cells by negatively regulating the Wnt/β-catenin signaling pathway.

Investigation of Drug-induced Lung Injury for the Development of a Novel Therapeutic Approach

The development of serious lung diseases, such as pulmonary fibrosis, is associated with several drugs. A recent study has shown that the epithelial-mesenchymal transition (EMT) plays an essential role in the development of pulmonary fibrosis. However, the mechanisms underlying drug-induced EMT in alveolar epithelial cells have not been characterized. The present study showed that methotrexate (MTX), a drug known to cause lung injury, induced EMT-like phenotypic changes in an A549 cell model of the alveolar epithelium. We also found that the transforming growth factor (TGF)-β1-mediated signaling pathway was associated with MTX-induced EMT. In addition, our results showed that certain secreted factors and microRNAs, a class of small noncoding RNAs, may be involved in MTX-induced EMT. The effects of COA-Cl, a novel synthetic small molecule, on TGF-β1-induced EMT were evaluated to determine the therapeutic potential of COA-Cl against drug-induced lung injury. COA-Cl significantly suppressed TGF-β1-induced EMT-like phenotypic changes, as evidenced by the inhibition of EMT-related transcription factors. Furthermore, MTX-induced EMT was completely suppressed by co-treatment with folic acid. Thus, these compounds may be promising therapeutic agents against drug-induced lung injury. In conclusion, the present study elucidated mechanisms underlying drug-induced EMT and highlighted a potential novel therapeutic approach to drug-induced lung diseases.

Anlotinib attenuated bleomycin-induced pulmonary fibrosis via the TGF-β1 signalling pathway.

Anlotinib hydrochloride (AL3818) is a novel multitarget tyrosine kinase inhibitor which has the same targets as nintedanib, an effective drug has been approved for the treatment of idiopathic pulmonary fibrosis. Here, we examined whether anlotinib could also attenuate bleomycin-induced pulmonary fibrosis in mice and explored the antifibrosis mechanism. We have evaluated the effect of anlotinib on bleomycin-induced pulmonary fibrosis in mice. Inflammatory cytokines in alveolar lavage fluid including IL-1β, IL-4, IL-6 and TNF-α were determined by ELISA. Biomarkers of oxidative stress were measured by corresponding kit. Histopathologic examination was analysed by H&E staining and immunohistochemistry. In vitro, we investigated whether anlotinib inhibited TGFβ/Smad3 and non-Smad pathways by luciferase assay or Western blotting. We also evaluated whether anlotinib inhibited TGF-β1-induced epithelial-mesenchymal transition (EMT) and promoted myofibroblast apoptosis in order to explore the possible molecular mechanism. The results indicated that anlotinib treatment remarkably attenuated inflammation, oxidative stress and pulmonary fibrosis in mouse lungs. Anlotinib could inhibit the TGF-β1 signalling pathway. Additionally, anlotinib not only profoundly inhibited TGF-β1-induced EMT in alveolar epithelial cells, but also simultaneously reduced the proliferation and promoted the apoptosis in fibroblasts. In summary, the results suggest that anlotinib-mediated suppression of pulmonary fibrosis is related to the inhibition of TGF-β1 signalling pathway.

Autophagy inhibition-mediated epithelial\u2013mesenchymal transition augments local myofibroblast differentiation in pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF), the prototypic progressive fibrotic interstitial lung disease, is thought to be a consequence of repetitive micro-injuries to an ageing, susceptible alveolar epithelium. Ageing is a risk factor for IPF and incidence has been demonstrated to increase with age. Decreased (macro)autophagy with age has been reported extensively in a variety of systems and diseases, including IPF. However, it is undetermined whether the role of faulty autophagy is causal or coincidental in the context of IPF. Here, we report that in alveolar epithelial cells inhibition of autophagy promotes epithelial–mesenchymal transition (EMT), a process implicated in embryonic development, wound healing, cancer metastasis and fibrosis. We further demonstrate that this is attained, at least in part, by increased p62/SQSTM1 expression that promotes p65/RELA mediated-transactivation of an EMT transcription factor, Snail2 (SNAI2), which not only controls EMT but also regulates the production of locally acting profibrogenic mediators. Our data suggest that reduced autophagy induces EMT of alveolar epithelial cells and can contribute to fibrosis via aberrant epithelial–fibroblast crosstalk.

Exogenous angiotensin (1-7) directly inhibits epithelial-mesenchymal transformation induced by transforming growth factor-β1 in alveolar epithelial cells

Accumulating evidence indicates that angiotensin (1-7) [Ang-(1-7)] protects against idiopathic pulmonary fibrosis (IPF) in animal experiments. However, whether Ang-(1-7) effectively inhibits epithelial-mesenchymal transition (EMT) induced by transforming growth factor-β1 (TGF-β1) remains unclear. The aim of this study is to examine the eff ;ects of Ang-(1-7) on TGF-β1-induced EMT in human alveolar epithelial cells. We found that angiotensin-converting enzyme 2 (ACE2) /Ang-(1-7)/MasR were decreased in the lungs of mice with IPF induced by bleomycin, and were negatively correlated with Tgfb1 mRNA expression. In vitro, our data showed that exogenous Ang-(1-7) restored the expression of E-cadherin and decreased the expressions of α-SMA and Vimentin induced by TGF-β1 in A549 cells. Ang-(1-7) also reduced TGF-β1-induced migration and synthesis of the extracellular matrix, such as collagen Ⅰ and collagen Ⅲ. Mechanistically, we observed that Ang-(1-7) directly inhibited TGF-β1-induced phosphorylation of Smad2 and Smad3, and suppressed the expression of the downstream target gene of TGF-β1-Smad signaling, including ZEB1, ZEB2, TWIST, and SNAIL1. Additionally, phosphorylation of mTOR induced by TGF-β1 also been suppressed by Ang-(1-7) treatment in A549 cells. Interestingly, we found that TGF-β1 strongly suppressed the expression of ACE2 in A549 cells through inhibiting SIRT1. In conclusion, our findings indicate that Ang-(1-7) directly inhibits TGF-β1-induced EMT in alveolar epithelial cells via disruption of TGF-β1-Smad signaling pathway, contributing to the protective effect against IPF.

Gas6 Prevents Epithelial-Mesenchymal Transition in Alveolar Epithelial Cells via Production of PGE2, PGD2 and Their Receptors

The epithelial-mesenchymal transition (EMT) is important in organ fibrosis. We hypothesized that growth arrest-specific protein 6 (Gas6) and its underlying mechanisms play roles in the prevention of EMT in alveolar epithelial cells (ECs). In this study, to determine whether Gas6 prevents TGF-β1-induced EMT in LA-4 and primary alveolar type II ECs, real-time PCR and immunoblotting in cell lysates and ELISA in culture supernatants were performed. Migration and invasion assays were performed using Transwell chambers. Pretreatment of ECs with Gas6 inhibited TGF-β1-induced EMT based on cell morphology, changes in EMT marker expression, and induction of EMT-activating transcription factors. Gas6 enhanced the levels of cyclooxygenase-2 (COX-2)-derived prostaglandin E2 (PGE2) and PGD2 as well as of their receptors. COX-2 inhibitors and antagonists of PGE2 and PGD2 receptors reversed the inhibition of TGF-β1-induced EMT, migration, and invasion by Gas6. Moreover, knockdown of Axl or Mer reversed the enhancement of PGE2 and PGD2 and suppression of EMT, migration and invasion by Gas6. Our data suggest Gas6-Axl or -Mer signalling events may reprogram ECs to resist EMT via the production of PGE2, PGD2, and their receptors.