4088

SMAD3

Idiopathic pulmonary fibrosis is characterized by myofibroblast accumulation, extracellular matrix (ECM) remodeling, and excessive collagen deposition. ECM-producing myofibroblasts may originate from epithelial cells through epithelial to mesenchymal transition (EMT). TGF-beta1 is an inducer of EMT in pulmonary epithelial cells in vitro and in vivo, though the mechanisms are unclear. We hypothesized that TGF-beta1 induced EMT through Smad-dependent and -independent processes. To test this hypothesis, we studied the roles and mechanisms of TGF-beta1-induced Smad and p38 mitogen-activated protein kinase (MAPK) signaling in EMT-related changes in pulmonary epithelial cells. Exposure of pulmonary epithelial 1HAEo(-) cells to TGF-beta1 resulted in morphological and molecular changes of EMT over a 96-h period; loss of cell-cell contact, cell elongation, down-regulation of E-cadherin, up-regulation of fibronectin, and up-regulation of collagen I. Both Smad2/3 and p38 MAPK signaling pathways were activated by TGF-beta1. However, neither Smad2/3 nor p38 MAPK were required for the down-regulation of E-cadherin, yet p38 MAPK was associated with fibronectin up-regulation. Both Smad2/3 and p38 MAPK had a role in regulation of TGF-beta1-induced collagen expression. Furthermore, these data demonstrate that Smads and p38 MAPK differentially regulate EMT-related changes in pulmonary epithelial cells.CI - Copyright (c) 2010 Wiley-Liss, Inc.

The versatile cytokine transforming growth factor beta (TGF-beta) regulates cellular growth, differentiation, and migration during embryonic development and adult tissue homeostasis. Activation of TGF-beta receptors leads to phosphorylation of Smad2 and Smad3, which oligomerize with Smad4 and accumulate in the nucleus where they recognize gene regulatory regions and orchestrate transcription. Termination of Smad-activated transcription involves Smad dephosphorylation, nuclear export, or ubiquitin-mediated degradation. In an unbiased proteomic screen, we identified poly(ADP-ribose) polymerase-1 (PARP-1) as a Smad-interacting partner. PARP-1 dissociates Smad complexes from DNA by ADP-ribosylating Smad3 and Smad4, which attenuates Smad-specific gene responses and TGF-beta-induced epithelial-mesenchymal transition. Thus, our results identify ADP-ribosylation of Smad proteins by PARP-1 as a key step in controlling the strength and duration of Smad-mediated transcription.CI - Copyright (c) 2010 Elsevier Inc. All rights reserved.

epithelial-mesenchymal transition (EMT) was shown to confer tumor cells with abilities essential for metastasis, including migratory phenotype, invasiveness, resistance to apoptosis, evading immune surveillance, and tumor stem cell traits. Therefore, inhibition of EMT can be an important therapeutic strategy to inhibit tumor metastasis. Here, we show that activation of peroxisome proliferator-activated receptor gamma (PPAR-gamma) inhibits transforming growth factor beta (TGF-beta)-induced EMT in lung cancer cells and prevents metastasis by antagonizing Smad3 function. Activation of PPAR-gamma by synthetic ligands (troglitazone and rosiglitazone) or by a constitutively active form of PPAR-gamma prevents TGF-beta-induced loss of E-cadherin expression and inhibits the induction of mesenchymal markers (vimentin, N-cadherin, fibronectin) and matrix metalloproteases. Consistently, activation of PPAR-gamma also inhibited EMT-induced migration and invasion of lung cancer cells. Furthermore, effects of PPAR-gamma ligands were attenuated by siRNA-mediated knockdown of PPAR-gamma, indicating that the ligand-induced responses are PPAR-gamma dependent. Selective knockdown of Smad2 and Smad3 by siRNA showed that TGF-beta-induced EMT is Smad3 dependent in lung cancer cells. Activation of PPAR-gamma inhibits TGF-beta-induced Smad transcriptional activity but had no effect on the phosphorylation or nuclear translocation of Smads. Consistently, PPAR-gamma activation prevented TGF-beta-induced transcriptional repression of E-cadherin promoter and inhibited transcriptional activation of N-cadherin promoter. Finally, treatment of mice with troglitazone or knockdown of Smad3 in tumor cells significantly inhibited TGF-beta-induced experimental metastasis in SCID-Beige mice. Together, with the low toxicity profile of PPAR-gamma ligands, our data show that these ligands may serve as potential therapeutic agents to inhibit metastasis.CI - (c)2010 AACR.

Previous studies demonstrate that response gene to complement 32 (RGC-32) mediates transforming growth factor-beta(1)-induced epithelial-mesenchymal transition (EMT) of human renal proximal tubular cells. However, the mechanisms underlying RGC-32 function remain largely unknown. In the present study, we found that RGC-32 function in EMT is associated with Smad3. Coexpression of RGC-32 and Smad3, but not Smad2, induces a higher mesenchymal marker alpha-smooth muscle actin (alpha-SMA) protein expression as compared with RGC-32 or Smad3 alone, while knockdown of Smad3 using short hairpin interfering RNA blocks RGC-32-induced alpha-SMA expression. These data suggest that RGC-32 interacts with Smad3, but not Smad2, in the regulation of EMT. In addition to alpha-SMA, RGC-32 and Smad3 also synergistically activate the expression of extracellular matrix protein fibronectin and downregulate the epithelial marker E-cadherin. RGC-32 colocalizes with Smad3 in the nuclei of renal proximal tubular cells. Coimmunoprecipitation assays showed that Smad3, but not Smad2, physically interacts with RGC-32 in renal proximal tubular cells. Mechanistically, RGC-32 and Smad3 coordinate the induction of EMT by regulating the EMT regulators Slug and Snail. Taken together, our data demonstrate for the first time that RGC-32 interacts with Smad3 to mediate the EMT of human renal proximal tubular cells.

In pancreatic ductal adenocarcinoma (PDAC), features of epithelial-mesenchymal transition (EMT) are often seen in tumor tissue, and such features correlate with poor prognosis. Solitary infiltration of tumor cells represents a morphological phenotype of EMT, and we previously reported that a high degree of solitary cell infiltration correlates with EMT-like features, including reduced E-cadherin and elevated vimentin levels. Using solitary cell infiltration to evaluate the degree of EMT, gene-expression profiling of 12 PDAC xenografts was performed, and SMAD3 was identified as an EMT-related gene. Immunohistochemistry using clinical specimens (n=113) showed that SMAD3 accumulated in the nuclei of tumor cells, but was not detected in most epithelial cells in the pancreatic duct. Moreover, SMAD3 upregulation correlated with malignant characteristics, such as higher tumor grade and lymph node metastasis, as well as with EMT-like features. SMAD4, which plays a key role in transforming growth factor-beta (TGF-beta) signaling, is inactivated in approximately half of PDAC cases. In this study, the nuclear accumulation of SMAD3 was immunohistochemically detected even in SMAD4-negative cases. SMAD3 knockdown resulted in upregulated E-cadherin, downregulated vimentin, and reduced cell motility in pancreatic cancer cells regardless of SMAD4 status. In addition, TGF-beta-treatment resulted in EMT induction in cells carrying wild-type SMAD4, and EMT was suppressed by SMAD3 knockdown. Patients with upregulated SMAD3 and a high degree of solitary cell infiltration had shorter times to recurrence and shorter survival times after surgery, and multivariate analysis showed that both factors were independent prognostic factors linked to unfavorable outcomes. These findings suggest that SMAD3 in PDAC is involved in the promotion of malignant potential through EMT induction in tumor cells regardless of SMAD4 status and serves as a potential biomarker of poor prognosis.

epithelial-mesenchymal transition (EMT) of retinal pigment epithelium (RPE) cells is a major pathologic change in the development of proliferative vitreoretinopathy (PVR), which leads to severe visual impairment. ERK1/2 pathway has been reported to play a key role in the carcinogenesis, cancer metastasis, and multiple fibrotic diseases. We hypothesized that ERK1/2 signaling could cross-interact with transforming growth factor beta2 (TGFbeta2)/Smad and Notch signaling pathways in the regulation of EMT in RPE cells. Here, we demonstrated that ERK1/2 signaling was activated in TGFbeta2-induced EMT in human RPE cells, while blockade of the canonical TGFbeta2/Smad2/3 signaling with SB431542 could not inhibit TGFbeta2-induced the activation of ERK1/2. Meanwhile, blockade of ERK1/2 signaling with a specific MEK/ERK1/2 inhibitor U0126 strongly prevented TGFbeta2-induced the downregulation of P-cadherin, and the upregulation of alpha-SMA, collagen type IV, N-cadherin and fibronectin in RPE cells. In addition, we also identified that blockade of ERK1/2 signaling could inhibit not only the canonical TGFbeta/Smad signaling, but also the Jagged/Notch pathway. Finally, we found that blockade of Notch pathway with a specific inhibitor DAPT could inhibit TGFbeta2-induced the activation of ERK1/2 pathway conversely. Therefore, our study provides evidence that ERK1/2 signaling can cross-interact with the canonical TGFbeta/Smad and the Jagged/Notch signaling pathways in RPE cells EMT. ERK1/2 inhibitor may have therapeutic value in the prevention and treatment of PVR and other fibrotic diseases.

TGF-beta/Smad signaling induces epithelial-mesenchymal transition (EMT) and tumor metastasis. As essential mediators in this pathway, Smad2 and Smad3 have been extensively studied and found to promote EMT and the subsequent mobility as well as invasiveness of lung cancer cells. In the present study, we determined that miR-136 directly targeted Smad2 and Smad3, leading to reduced migration and invasiveness of lung adenocarcinoma (ADC) cell lines, accompanied by increased epithelial markers as well as decreased mesenchymal markers. Moreover, ectopic expression of either Smad2 or Smad3 partially restored the malignant phenotype of ADC cells overexpressing miR-136. Taken together, our data demonstrate that miR-136 may play a tumor-suppressive role by repressing EMT and prometastatic traits via targeting Smad2 and Smad3. The potent antimetastasis property of miR-136 and its multitarget mechanism provide new insights in developing novel therapeutic approaches.

Metastasis is the major cause for the high mortality rate of pancreatic cancer. Human embryonic stem cell (hESC) associated genes frequently correlate with malignant disease progression. Recent studies have demonstrated that the embryonic protein Nodal, which plays a critical role during embryonic development, is re-expressed in several types of tumors and promotes cancers progression. However, little is known about the role of Nodal in pancreatic cancer. Here, we show that Nodal expression is upregulated in human pancreatic cancer tissues. Moreover, Nodal expression levels correlate well with the grade of pancreatic cancer differentiation. In addition, we present clear evidence that Nodal induces signal transduction through the Smad2/3-dependent pathway in vitro. Furthermore, we show that Nodal promotes pancreatic cancer cell migration and invasion, induces epithelial-mesenchymal transition (EMT) and enhances the expression of matrix metalloproteinase-2 (MMP2) and CXC chemokine receptor 4 (CXCR4). Using an in vivo liver metastasis model of pancreatic cancer, we observed that blocking Nodal signaling activity with the small-molecule inhibitor SB431542 decreases the number and size of liver metastases. Taken together, our results suggest that Nodal overexpression induces a metastatic phenotype in pancreatic cancer cells, and that targeting Nodal signaling may be a promising therapeutic strategy for pancreatic cancer.

BACKGROUND: The epithelial-mesenchymal transition (EMT) has been associated with the acquisition of migration, invasiveness, and metastasis traits. During tumor progression, EMT can be induced by transforming growth factor-beta (TGF-beta) signal that epithelial cells receive from their microenvironment. However, the master regulatory controls on TGF-beta-EMT axis are not understood. METHODS: The protein expression in human specimens was measured by immunohistochemical staining. E74-like factor 5 (Elf5) was silenced by short interfering RNAs in LNCaP cells and stably overexpressed by HA-tagged Elf5 cDNAs in 22Rv1 cells. These cells were used to study migration and anchorage-independent growth. RESULTS: Our data reveal that Elf5 results in the failure of mesenchymal morphogenesis, upregulation of EMT markers, spheres formation, and migration in the presence of TGF-beta. Furthermore, Elf5 blocks TGF-beta signaling, through decreasing drosophila mothers against decapentaplegic protein (SMAD3) activation by binding to it, one of the major effector of TGF-beta-induced EMT. Moreover, Elf5 can serve as a prognostic marker of metastasis-free survival in patients with TGF-beta-positive prostate cancer. CONCLUSIONS: Elf5 expression is inversely correlated with EMT. Elf5 inhibits TGF-beta-driven EMT via repressing SMAD3 phosphorylation in prostate cancer cells. In addition, Elf5 can be used as a biomarker of metastasis-free survival in patients with TGF-beta-positive prostate cancer.CI - (c) 2015 Wiley Periodicals, Inc.

epithelial-mesenchymal transition (EMT) is a key event in cancer metastasis, which confers cancer cells with increased motility and invasiveness, and EMT is characterized by loss of epithelial marker E-cadherin and gain of mesenchymal marker N-cadherin. Transforming growth factor-beta (TGF-beta) signaling is a crucial inducer of EMT in various types of cancer. Ski is an important negative regulator of TGF-beta signaling, which interacts with SMADs to repress TGF-beta signaling activity. Although there is accumulating evidence that Ski functions as a promoter or suppressor in human types of cancer, the molecular mechanisms by which Ski affects TGF-beta-induced EMT and invasion in non-small cell lung cancer (NSCLC) are not largely elucidated. In the present study, we investigated the mechanistic role of Ski in NSCLC metastasis. Ski was significantly reduced in metastatic NSCLC cells or tissues when compared with non-metastatic NSCLC cells or tissues. Moreover, following TGF-beta stimulation Ski-silenced A549 cells had more significant features of EMT and a higher invasive activity when compared with A549 cells overexpressing Ski. Mechanistically, Ski-silenced and overexpressed A549 cells showed an increase and a reduction in the SMAD3 phosphorylation level, respectively. This was supported by plasminogen activator inhibitor-1 (PAI-1) promoter activity obtained in Ski-silenced and overexpressed A549 cells. However, after treatment of SIS3 (inhibitor of SMAD3 phosphorylation) followed by TGF-beta1 stimulation, we did not observe any effect of Ski on TGF-beta-induced EMT, and invasion in Ski-silenced and overexpressed A549 cells. In conclusion, our findings suggest that Ski represses TGF-beta-induced EMT and invasion by inhibiting SMAD-dependent signaling in NSCLC.

epithelial-mesenchymal transition (EMT) is associated with the metastasis and poor prognosis of cervical cancer. However, the underlying mechanisms are poorly defined. In the present study, we investigated whether Twist plays a direct role in human cervical cancer using immunohistochemical and western blot analyses. Immunohistochemical analysis revealed that Twist is highly expressed in cervical cancer, which correlates with poor tumor pathological differentiation or lymph node metastasis (P<0.05). Depletion of Twist by stable shRNA-mediated knockdown decreased the migratory ability of cancer cell lines in vitro. Suppression or overexpression of Twist also resulted in an altered expression of the molecular mediators of EMT. Furthermore, exogenous TGF-beta promoted EMT by upregulating the expression of Twist through the TGF-beta/Smad3 pathway, and this effect was eliminated by Twist depletion in cancer cells as demonstrated in the in vitro study. The use of in vivo models revealed a decreased tumor proliferation potential in Twist-depleted cancer cells. The results suggested a novel function for Twist in the promotion of EMT via TGF-beta/Smad3 signaling pathway. Thus, Twist constitutes a potential therapeutic target in human cervical cancer.

epithelial-mesenchymal transition (EMT) is a normal cell differentiation event during development and contributes pathologically to carcinoma and fibrosis progression. EMT often associates with increased transforming growth factor-beta (TGF-beta) signaling, and TGF-beta drives EMT, in part through Smad-mediated reprogramming of gene expression. TGF-beta also activates the Erk MAPK pathway through recruitment and Tyr phosphorylation of the adaptor protein ShcA by the activated TGF-beta type I receptor. We found that ShcA protects the epithelial integrity of nontransformed cells against EMT by repressing TGF-beta-induced, Smad-mediated gene expression. p52ShcA competed with Smad3 for TGF-beta receptor binding, and down-regulation of ShcA expression enhanced autocrine TGF-beta/Smad signaling and target gene expression, whereas increased p52ShcA expression resulted in decreased Smad3 binding to the TGF-beta receptor, decreased Smad3 activation, and increased Erk MAPK and Akt signaling. Furthermore, p52ShcA sequestered TGF-beta receptor complexes to caveolin-associated membrane compartments, and reducing ShcA expression enhanced the receptor localization in clathrin-associated membrane compartments that enable Smad activation. Consequently, silencing ShcA expression induced EMT, with increased cell migration, invasion, and dissemination, and increased stem cell generation and mammosphere formation, dependent upon autocrine TGF-beta signaling. These findings position ShcA as a determinant of the epithelial phenotype by repressing TGF-beta-induced Smad activation through differential partitioning of receptor complexes at the cell surface.

BACKGROUND/AIMS: Transforming growth factor beta (TGF-beta) plays a major role in tumorigenesis. MicroRNA-181b (miRNA-181b) is a multifaceted miRNA that has been implicated in many cellular processes such as cell fate determination and cellular invasion. This study aimed to confirm the relationship of miRNA-181b and the TGF-beta-Smad2/3/4 pathway with the induction of the epithelial-to-mesenchymal transition (EMT) in gastric cancer. METHODS: This study investigated the ability of TGF-beta to induce migration by wound healing and transwell invasion assays in human gastric cancer cell lines. miRNA expression was altered using miRNA-181b mimic and inhibitor in the same system. expression of miRNA-181b, the hypothetical target gene Timp3 and EMT-related markers were analyzed by real-time real-time quantitative RT-PCR. Immunoblotting was used to investigate the levels of phospho-Smad2 and Smad4. Dual-luciferase reporter assays were performed to confirm the direct binding of miRNA-181b to Timp3. RESULTS: miRNA-181b was significantly upregulated in response to TGF-beta treatment in gastric cancer cell lines. Overexpression of miR-181b mimic induced an in vitro EMT-like change to a phenotype similar to that following TGF-beta treatment alone and was reversed by miRNA-181b inhibitor. Inhibition of TGF-beta-Smad2/3 signaling with SD-208 significantly attenuated the upregulation of miRNA-181b. Knockdown of Smad4 in gastric cancer cells strongly attenuated the upregulation of miRNA-181b. Moreover, miR-181b was found to directly target the 3' untranslated region (3'UTR) of Timp3 mRNA affecting TGF-beta-induced EMT. CONCLUSIONS: Our results elucidate a novel mechanism through which the TGF-beta pathway regulates the EMT of gastric cancer cells by increasing the levels of miRNA-181b to target Timp3 via the Smad2/3/4-dependent pathway. These findings provide insights into the cellular and environmental factors regulating EMT, which may guide future studies on therapeutic strategies targeting these cells.CI - (c) 2016 The Author(s) Published by S. Karger AG, Basel.

Vasculogenic mimicry (VM) is a nonangiogenesis-dependent pathway that promotes tumor growth and disease progression. Nodal signaling has several vital roles in both embryo development and cancer progression. However, the effects of Nodal signaling on VM formation in breast cancer and its underlying mechanisms are ill-defined. We analyzed the relationship between Nodal signaling and VM formation in one hundred human breast cancer cases and the results showed that the expression of Nodal was significantly correlated with VM formation, tumor metastasis, differentiation grade, TNM stage and poor prognosis. Furthermore, up-regulation of Nodal expression promoted VM formation of breast cancer cells in vitro and in vivo. Knockdown of Nodal expression restrained VM formation. In addition, Nodal induced EMT and up-regulated the expression of Slug, Snail and c-Myc. We found that blocking the Smad2/3 pathway by administering SB431542 inhibited VM formation in breast cancer cell lines and xenografts. Taken together, Nodal signaling through the Smad2/3 pathway up-regulated Slug, Snail and c-Myc to induce EMT, thereby promoting VM formation. Our study suggests that the Nodal signaling pathway may serve as a therapeutic target to inhibit VM formation and improve prognosis in breast cancer patients.

Sec8 is one of the subunits of the exocyst, which is an evolutionarily conserved complex of eight proteins, comprising Sec3 (EXOC1), Sec5 (EXOC2), Sec6 (EXOC3), Sec8 (EXOC4), Sec10 (EXOC5), Sec15 (EXOC6), Exo70 (EXOC7), and Exo84 (EXOC8) subunits. Sec8 knockout mice embryos initiate gastrulation but are unable to progress beyond the primitive streak stage and die shortly. During embryonic development, the first epithelial-mesenchymal transition (EMT) event occurs at gastrulation. Sec8 may be involved in the early embryonic development through EMT. However, the function of Sec8 in EMT remains unclear. In the present study, it was found that Sec8 regulates N-cadherin expression by controlling Smad3 and Smad4 expression at the basal transcriptional level, thereby modulating cell migration and adhesion. Furthermore, Sec8 knockdown decreased CREB binding protein (CBP) expression at mRNA and protein levels. However, CBP knockdown did not affect Sec8 expression. These results indicated that Sec8 regulates N-cadherin expression by controlling Smad3 and Smad4 expression through CBP, thereby mediating the EMT.CI - Copyright A(c) 2016 Elsevier Inc. All rights reserved.

Atrial natriuretic peptide (ANP) is increasingly expressed on airway and inhibits pulmonary arterial remodeling. However, the role of ANP in remodeling of respiratory system is still unclear. The role of ANP on airway remodeling and the possible mechanism was explored in this study. Both human bronchial epithelial 16HBE-14o cells and alveolar epithelial A549 cells were stimulated by TGF-beta1, ANP, cGMP inhibitor, PKG inhibitor, and cGMP analogue. The expressions of epithelial markers, mesenchymal markers, and Smad3 were assessed by quantitative real-time PCR and western blotting. Immunohistochemical staining was employed to assess Smad3 expression once it was silenced by siRNA in 16HBE-14o or A549 cells. Our results showed that the mRNA and protein expressions of E-Cadherin were decreased, whereas alpha-SMA expressions were increased after induction by TGF-beta1 in 16HBE-14o and A549 cells. The E-Cadherin expressions were increased and alpha-SMA expressions were decreased after ANP stimulation. Inhibition of cGMP or PKG decreased E-Cadherin expression but increased alpha-SMA expression, which could be reversed by cGMP analogue. Moreover, the phosphorylated Smad3 expression was consistent with alpha-SMA expression. After smad3 was silenced, Smad3 was mostly expressed in cytoplasm instead of nucleus as non-silenced cells during epithelial-mesenchymal transition (EMT). In conclusion, ANP inhibits TGF-beta1-induced EMT in 16HBE-14o and A549 cells through cGMP/PKG signaling, by which it targets TGF-beta1/Smad3 via attenuating phosphorylation of Smad3. These findings suggest the potential of ANP in the treatment on pulmonary diseases with airway remodeling.CI - Copyright (c) 2017 Elsevier Inc. All rights reserved.

Cytosolic phospholipase A2alpha (cPLA2alpha), a key phospholipase that regulates lipid metabolism, plays an important role in tumor progression. In the present study of hepatocellular carcinoma (HCC), cPLA2alpha was overexpressed in highly metastatic HCC cell lines. Immunohistochemical staining showed increased levels of cPLA2alpha at the invasive edges of HCC, and a clinicopathological analysis of samples from 111 patients revealed that its expression level was linked with micro-vascular invasion and cirrhosis. Knockdown of cPLA2alpha inhibited migration, probably due to its role in actin polymerization. Overexpression of cPLA2alpha promoted cell migration and invasion. Based on the mechanistic analysis, our data suggested that cPLA2alpha mediate epidermal growth factor (EGF) induced epithelial-mesenchymal transition (EMT) through PI3K/AKT/ERK pathway. cPLA2alpha activity was required for the transforming growth factor-(TGF)-beta-induced EMT. However, cPLA2alpha inhibited Smad2/3 activation and promoted the activation of the PI3K/AKT/ERK pathway. A xenograft tumor transplant model confirmed the role of cPLA2alpha in HCC invasion and metastasis. Based on the mechanistic analysis, cPLA2alpha mediated both EGF- and TGF-beta-induced EMT, which are essential for HCC metastasis. cPLA2alpha is a potentially target for novel therapies of HCC.CI - Copyright (c) 2017 Elsevier B.V. All rights reserved.

As a Ca(2+) binding protein, calreticulin (CRT) has many functions and plays an important role in a variety of tumors. The role of CRT in TGF-beta1-induced EMT is unknown. In this study, we demonstrated in vitro that TGF-beta1-induced EMT elevated the expression of CRT in A549 lung cancer cells. Subsequently, we confirmed that overexpression CRT had no capacity to induce A549 cells EMT alone, but successfully enhanced TGF-beta1-induced-EMT. Furthermore, knockdown of CRT in A549 cells significantly suppressed changes of EMT marks expression induced by TGF-beta1. On treatment with TGF-beta1, overexpression of CRT could enhance the phosphorylation of both Smad2 and Smad3. Consistently, the knockdown of CRT by siRNA-CRT could inhibit Smad signaling pathway activated by TGF-beta1. These results indicated that CRT regulates EMT induced by TGF-beta1 through Smad signaling pathway. Finally, TGF-beta1-induced-EMT enhanced store-operated Ca(2+) influx in A549 cells. CRT knockdown was able to abolish the effect of TGF-beta1 on thapsigargin (TG) -induced Ca(2+) release, but had failed to reduce store-operated Ca(2+) influx. The alteration of intracellular Ca(2+) concentration by TG or BAPTA-AM was able to regulate EMT induced by TGF-beta1 through Smad signaling pathway. Together, these data identify that CRT regulates TGF-beta1-induced-EMT through modulating Smad signaling. Furthermore, TGF-beta1-induced-EMT is highly calcium-dependent, CRT was partly involved in it.CI - Copyright (c) 2017 Elsevier Ltd. All rights reserved.

Idiopathic pulmonary fibrosis (IPF) is a chronic and usually progressive lung disease and the epithelial-mesenchymal transition (EMT) may play an important role in the pathogenesis of pulmonary fibrosis. IL-17 is a proinflammatory cytokine which promotes EMT profiles in lung inflammatory diseases. In this study, we investigated the effect of IL-17 on EMT in alveolar epithelial cell line A549 and the role of TGFbeta1-Smad and ERK signaling pathways in the process. Morphological observation on the cells was performed under inverted microscope. The mRNA and protein expressions of E-cad and alpha-SMA were detected by quantitative RT-PCR and western blotting. The mRNA and protein expressions of TGF-beta1 were analyzed via quantitative RT-PCR and ELISA. expressions of Smad2/3, p-Smad2/3, ERK1/2, p-ERK1/2 and p-JNK were examined by western blotting. The results indicated that IL-17 can induce A549 cells to undergo morphological changes and phenotypic markers changes, such as down-regulated E-cad expression and up-regulated alpha-SMA expression. Additionally, IL-17 enhanced TGF-beta1 expression and stimulated Smad2/3 and ERK1/2 phosphorylation in A549 cells. However, there were no significant differences in the expression of phosphorylated JNK in A549 cells with or without IL-17 treatment. SB431542 or U0126 treated cells showed inhibited morphological changes and phenotypic markers expression, such as up-regulated E-cad expression and down-regulated alpha-SMA expression. In summary, our results suggest that IL-17 can induce A549 alveolar epithelial cells to undergo EMT via the TGF-beta1 mediated Smad2/3 and ERK1/2 activation.

Thymoquinone, a major ingredient of black seed oil (Nigella sativa), has been shown to exhibit anticancer capacity in various types of cancers. However, there are few studies concerning the correlation between thymoquinone and epithelial-to-mesenchymal transition (EMT) in prostate cancer. In the present study, we firstly found that thymoquinone showed antimetastatic capacity in prostate cancer DU145 and PC3 cells. Additionally, thymoquinone reversed EMT by increasing E-cadherin expression and decreasing vimentin and Slug expression in a concentration-dependent manner. Recent studies have shown that the transforming growth factor-beta (TGF-beta) signaling pathway may be associated with EMT. Intriguingly, the expression of TGF-beta, Smad2 and Smad3 at the mRNA and protein levels was notably reduced upon thymoquinone treatment in prostate cancer DU145 and PC3 cells. Subsequently, we confirmed that thymoquinone repressed metastasis and EMT of prostate cancer through downregulation of the TGF-beta/Smad2/3 signaling pathway, which may be partially reversed by TGF-beta overexpression. In summary, our findings demonstrated that thymoquinone suppressed the metastatic phenotype and reversed EMT of prostate cancer cells by negatively regulating the TGF-beta/Smad2/3 signaling pathway. These findings suggest that thymoquinone is a potential therapeutic agent against prostate cancer which functions by targeting TGF-beta.

Smad3 and STAT3 are intracellular molecules that transmit signals from plasma membrane receptors to the nucleus. Smad3 operates downstream of growth/differentiation factors that utilize activin receptor-like kinase (ALK)-4, 5, or 7, such as transforming growth factor-beta (TGF-beta), activin, and myostatin. STAT3 principally functions downstream of cytokines that exert their effects via gp130 and Janus family kinases, including interleukin-6 (IL-6), leukemia inhibitory factor (LIF), and oncostatin M. Accumulating evidence indicates that Smad3 and STAT3 engage in crosstalk in a highly context-dependent fashion, cooperating in some conditions while acting antagonistically each other in others. Here, we review the crosstalk between Smad3 and STAT3 in various biological contexts, including early tumorigenesis, epithelial-mesenchymal transition, fibrosis, and T cell differentiation.CI - (c) The Author 2017. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Pulmonary fibrosis (PF) is a chronic lung disease. The transforming growth factor-beta1 (TGF-beta1)/Smad3 signaling pathway plays an important role in the pathogenesis of pulmonary fibrosis. Bone marrow-derived mesenchymal stem cells (BMSCs) have been shown to be a modulator of the molecular aspects of the fibrosis pathway. However, it is still unknown as to whether the conditioned medium from BMSCs (BMSCs-CM) inhibits the epithelial-mesenchymal transition (EMT) process. This study confirmed the hypothesis that BMSCs-CM exerts an anti-fibrotic effect on human type II alveolar epithelial cells (A549) by suppressing the phosphorylation of Smad3. We used the A549 cells in vitro to detect morphological evidence of EMT by phase-contrast microscopy. These cells were randomly divided into 4 groups as follows: the control group, the TGF-beta1 group, the SIS3 (specific inhibitor of Smad3) group and the BMSCs-CM group. The immunofluorescence method was used to determined the location of E-cadherin (E-calcium mucins; E-cad), alpha-smooth muscle actin (alpha-SMA) and p-Smad3. The expression levels of E-cad, CK8, alpha-SMA, vimentin, p-Smad3, Snail1, collagen I (COLI) and collagen III (COLIII) were detected by western blot analysis. Following exposure to TGF-beta1, the A549 cells displayed a spindle-shaped fibroblast-like morphology. In accordance with these morphological changes, the expression levels of E-cad and CK8 were downregulated, while the expression levels of alpha-SMA and vimentin were upregulated. Along with this process, the expression levels of p-Smad3, Snail1, COLI and COLIII were increased. However, the cells in the BMSCs-CM group and SIS3 group exhibited a decrease in the levels of alpha-SMA and vimentin (which had been upregulated by TGF-beta1), and an increase in the levels of E-cad and CK8 expression (which had been downregulated by TGF-beta1). On the whole, these results indicated that BMSCs-CM suppressed the EMT which might be associated with TGF-beta1/Smad3. This study provides the theoretical basis for the research of the mechanisms responsible for pulmonary disease.

During epithelial-mesenchymal transition (EMT), reprogramming of gene expression is accompanied by histone modifications. Whether EMT-promoting signaling directs functional changes in histone methylation has not been established. We show here that the histone lysine methyltransferase SETDB1 represses EMT and that, during TGF-beta-induced EMT, cells attenuate SETDB1 expression to relieve this inhibition. SETDB1 also controls stem cell generation, cancer cell motility, invasion, metastatic dissemination, as well as sensitivity to certain cancer drugs. These functions may explain the correlation of breast cancer patient survival with SETDB1 expression. At the molecular level, TGF-beta induces SETDB1 recruitment by Smad3, to repress Smad3/4-activated transcription of SNAI1, encoding the EMT "master" transcription factor SNAIL1. Suppression of SNAIL1-mediated gene reprogramming by SETDB1 occurs through H3K9 methylation at the SNAI1 gene that represses its H3K9 acetylation imposed by activated Smad3/4 complexes. SETDB1 therefore defines a TGF-beta-regulated balance between histone methylation and acetylation that controls EMT.CI - (c) 2017 The Authors.

Pancreatic cancer is the fourth leading cause of cancer-associated mortality worldwide, with an overall 5-year survival rate <8%. We studied the therapeutic effect of itraconazole (ITZ), a commonly used broad-spectrum anti-fungal agent, in the treatment of pancreatic cancer, and to reveal the underlying anticancer mechanisms. Effects of ITZ on cell proliferation, apoptosis, invasion and migration were observed by MTT assays and colony formation assays, flow cytometry, wound scratch assays and transwell assays, respectively. Western blotting and immunofluorescence were performed to investigate the effect of ITZ on the epithelial to mesenchymal transition (EMT) of pancreatic cancer cells. Recombinant transforming growth factor-beta (TGF-beta) and TGF-beta neutralizing antibody were used to study the effect of ITZ on the TGF-beta/SMAD2/3 signaling. Transgenic engineered mice which harboring the spontaneous pancreatic cancer was applied to investigate the therapeutic role of ITZ in vivo. We report that ITZ inhibited the viability and induced apoptosis of pancreatic cancer cells. Furthermore, ITZ suppressed the invasion and migration of pancreatic cancer cells. We found that ITZ treatment was efficient in suppressing EMT and that the effect of ITZ was partially mediated by impaired TGF-beta/SMAD2/3 signaling. The role of TGF-beta/SMAD2/3 signaling in mediating the effect of ITZ was confirmed based on the results that recombinant TGF-beta induced, but the TGF-beta neutralizing antibody inhibited EMT as well as the invasion and migration of pancreatic cancer cells. Also, the anticancer effect of ITZ could be partially reversed by recombinant TGF-beta. Furthermore, treatment with ITZ suppressed growth of tumor in vivo. Taken together, we suggest that ITZ may potentially serve as a new chemotherapeutic agent for the treatment of pancreatic cancer.

Activation of metastatic reprogramming is critical for tumour metastasis. However, more detailed knowledge of the underlying mechanism is needed to enable targeted intervention. Here, we show that paraspeckle component 1 (PSPC1), identified in an aberrant 13q12.11 locus, is upregulated and associated with poor survival in patients with cancer. PSPC1 promotes tumorigenesis, epithelial-to-mesenchymal transition (EMT), stemness and metastasis in multiple cell types and in spontaneous mouse cancer models. PSPC1 is the master activator for transcription factors of EMT and stemness and accompanies c-Myc activation to facilitate tumour growth. PSPC1 increases transforming growth factor-beta1 (TGF-beta1) secretion through an interaction with phosphorylated and nuclear Smad2/3 to potentiate TGF-beta1 autocrine signalling. Moreover, PSPC1 acts as a contextual determinant of the TGF-beta1 pro-metastatic switch to alter Smad2/3 binding preference from tumour-suppressor to pro-metastatic genes. Having validated the PSPC1-Smads-TGF-beta1 axis in various cancers, we conclude that PSPC1 is a master activator of pro-metastatic switches and a potential target for anti-metastasis drugs.

Lens epithelial cells undergo epithelial-mesenchymal transition (EMT) after injury as in cataract extraction, leading to fibrosis of the lens capsule. Fibrosis of the anterior capsule can be modeled in the mouse by capsular injury in the lens, which results in EMT of the lens epithelium and subsequent deposition of extracellular matrix without contamination of other cell types from outside the lens. We have previously shown that signaling via Smad3, a key signal-transducing element downstream of transforming growth factor (TGF)-beta and activin receptors, is activated in lens epithelial cells by 12 hours after injury and that this Smad3 activation is blocked by administration of a TGF-beta 2-neutralizing antibody in mice. We now show that EMT of primary lens epithelial cells in vitro depends on TGF-beta expression and that injury-induced EMT in vivo depends, more specifically, on signaling via Smad3. Loss of Smad3 in mice blocks both morphological changes of lens epithelium to a mesenchymal phenotype and expression of the EMT markers snail, alpha-smooth muscle actin, lumican, and type I collagen in response to injury in vivo or to exposure to exogenous TGF-beta in organ culture. The results suggest that blocking the Smad3 pathway might be beneficial in inhibiting capsular fibrosis after injury and/or surgery.

We previously reported that osteopontin (OPN), a matrix structural glycophosphoprotein, is upregulated in the injured mouse lens prior to the epithelial-mesenchymal transition (EMT). Here, we investigated the role of this protein in EMT of the lens epithelium during wound healing. The crystalline lens was injured by needle puncture in OPN-null (KO, n=40) and wild-type (WT, n=40) mice. The animals were killed at day 1, 2, 5, and 10 postinjury. Immunohistochemistry was employed to detect alpha-smooth muscle action (alphaSMA), a marker of EMT, collagen type I, transforming growth factor beta1 (TGFbeta1), TGFbeta2, and phospho-Smad2/3. Cell proliferation was assayed by examining uptake of bromodeoxyuridine (BrdU). The results showed that injury-induced EMT of mouse lens epithelium, as evaluated by histology, expression pattern of alphaSMA and collagen I, was altered in the absence of OPN with reduced phospho-Smad2/3 signaling. Upregulation of TGFbeta1 and TGFbeta2 in the epithelium was also inhibited. Cell proliferation was more active in KO mice as compared with WT mice at day 1 and 2, but not at day 5 and 10. An in vitro experiment shows OPN facilitates cell adhesion of lens epithelial cell line. OPN is required for activation of Smad2/3 signal in an injured lens epithelium and lens cell EMT.

Transforming growth factor (TGF)-beta1 is a key mediator of lung remodeling and fibrosis. epithelial cells are both a source of and can respond to TGF-beta1 with epithelial-to-mesenchymal transition (EMT). We recently determined that TGF-beta1-induced EMT in lung epithelial cells requires the presence of c-Jun N-terminal kinase (JNK) 1. Because TGF-beta1 signals via Smad complexes, the goal of the present study was to determine the impact of JNK1 on phosphorylation of Smad3 and Smad3-dependent transcriptional responses in lung epithelial cells. Evaluation of JNK1-deficient lung epithelial cells demonstrated that TGF-beta1-induced terminal phosphorylation of Smad3 was similar, whereas phosphorylation of mitogen-activated protein kinase sites in the linker regions of Smad3 was diminished, in JNK1-deficient cells compared with wild-type cells. In comparison to wild-type Smad3, expression of a mutant Smad3 in which linker mitogen-activated protein kinase sites were ablated caused a marked attenuation in JNK1 or TGF-beta1-induced Smad-binding element transcriptional activity, and expression of plasminogen activator inhibitor-1, fibronectin-1, high-mobility group A2, CArG box-binding factor-A, and fibroblast-specific protein-1, genes critical in the process of EMT. JNK1 enhanced the interaction between Smad3 and Smad4, which depended on linker phosphorylation of Smad3. Conversely, Smad3 with phosphomimetic mutations in the linker domain further enhanced EMT-related genes and proteins, even in the absence of JNK1. Finally, we demonstrated a TGF-beta1-induced interaction between Smad3 and JNK1. Collectively, these results demonstrate that Smad3 phosphorylation in the linker region and Smad transcriptional activity are directly or indirectly controlled by JNK1, and provide a putative mechanism whereby JNK1 promotes TGF-beta1-induced EMT.

PURPOSE: To develop a new animal model of anterior subcapsular cataract formation by topical application of alkali to the eye and to examine the role of Transforming growth factorbeta/Smad3 (TGFbeta/Smad3) signaling in the formation of this cataract model. METHODS: Under anesthesia, one eye of adult Wistar rats (n=142) was subjected to alkali burn by topical application of 1 N NaOH. The eye was then histologically examined at specific time intervals. Immunohistochemistry with a battery of antibodies was carried out to examine the epithelial-mesenchymal transition (EMT) in lens epithelium. Enzyme immunoassay was employed to determine the level of growth factors in aqueous humor and lens tissue. Smad3-null mice were also used to examine the role of Smad3 signaling in cataractogenesis in this model. RESULTS: Two days post-burn of the ocular surface, lens epithelium underwent EMT as evidenced by the upregulation of Snail and alpha-smooth muscle actin and formed a multilayer of cells beneath the capsule. Smad signaling was found to be activated in EMT-type lens cells. The majority of myofibroblast-type lens cells expressed proliferative cell nuclear antigen (PCNA). The total amount of active TGFbeta2, total TGFbeta2, and Fibroblast growth factor 2 (FGF2) increased in the aqueous humor and lens. Loss of Smad3 attenuated, but did not completely abolish, EMT in the lens epithelium. CONCLUSIONS: Topical alkali treatment of the ocular surface readily induces an EMT-type anterior subcapsular cataract. Smad3 signaling is involved, but not required, for achievement of EMT in the lens epithelium in this cataract model.

PURPOSE: Extracellular matrix (ECM) accumulates during the development of posterior capsule opacification (PCO). Vitronectin, an ECM component that is generally prominent in wound healing, has been detected in PCO specimens. Here we set out to investigate the distribution of vitronectin in the lens and determine how it, and other ECM components, influence the lens epithelial phenotype. METHODS: Rat lens epithelial explants were cultured on vitronectin, fibronectin, and laminin substrata. Explants were monitored for cell migration and the appearance of markers for epithelial mesenchymal transition (EMT), using phase contrast microscopy and immunohistochemistry, respectively. Explants were also monitored for evidence of Smad signaling. Vitronectin expression was analyzed in embryonic and postnatal rodent lens development by immunohistochemistry, western blotting, and in situ hybridization. RESULTS: Vitronectin, like fibronectin and laminin, provided a good substratum for cellular attachment and migration. However, in the case of vitronectin and fibronectin, this was accompanied by a major phenotypic change. On either vitronectin or fibronectin, but not laminin, most of the cells became elongated, spindle-shaped and were strongly reactive for filamentous alpha-smooth muscle actin. In these respects this transition was typical of the well known TGFbeta-induced EMT. In explants cultured on vitronectin and fibronectin, but not laminin, cell nuclei showed prominent reactivity for Smad 2/3. Vitronectin was also shown to be expressed during embryonic and postnatal development. Initially mRNA and protein were detected in all lens cells, however as development progressed, expression became restricted to cells of the epithelium and transition zone. CONCLUSIONS: The results clearly show that lens cell engagement with a vitronectin or a fibronectin, but not laminin, substratum has a potent EMT promoting effect and that Smad 2/3 signaling is involved. Thus when considering strategies to slow or prevent PCO, these results highlight the need to take into account ECM molecules such as vitronectin that have the capacity to promote EMT.

AIM: To investigate the mechanism by which Qinggan Huoxue Recipe (QGHXR) inhibits epithelial-to-mesenchymal transition (EMT) in rats with alcoholic liver fibrosis (ALF). METHODS: A total of 75 male SD rats were used to induce ALF. Serum biochemical indicators, including alanine aminotransferase, aspartate aminotransferase, laminin and hyaluronidase, were measured. Liver histopathological changes were evaluated using hematoxylin-eosin and Sirius red staining. EMT was examined by analyzing the expression of the epithelial marker E-cadherin and the mesenchymal markers vimentin and fibronectin using RT-PCR and Western blot. The inhibitory effect of QGHXR on EMT markers, as well as its effect on molecules associated with the transforming growth factor (TGF)-beta1/Smad signaling pathway, including TGF-beta1, Smad3, snail, occludin, ZO-1 and claudin, was also examined. RESULTS: Compared with normal control rats, ALF rats exhibited a decrease in E-cadherin levels (mRNA: ALF 0.16 +/- 0.05 vs control 1.00 +/- 0.08; protein: ALF 0.09 +/- 0.05 vs control 0.70 +/- 0.17, P < 0.01) and an increase in vimentin and fibronectin levels (mRNA: 11.43 +/- 0.39 vs 1.00 +/- 0.19 and 9.91 +/- 0.34 vs 1.00 +/- 0.44, respectively, P < 0.01; protein: 1.13 +/- 0.42 vs 0.09 +/- 0.03 and 1.16 +/- 0.43 vs 0.09 +/- 0.00, respectively, P < 0.01). This indicates that EMT occurred in ALF rats. In addition, the TGF-beta1/Smad signaling pathway was activated in ALF rats, as evidenced by the increase in TGF-beta1 and snail levels (mRNA: 1.76 +/- 0.12 vs 1.00 +/- 0.05 and 6.98 +/- 0.41 vs 1.00 +/- 0.10, respectively, P < 0.01; protein: 1.43 +/- 0.05 vs 0.12 +/- 0.03 and 1.07 +/- 0.29 vs 0.07 +/- 0.02, respectively, P < 0.01) and the decrease in Smad3 levels (mRNA: 0.05 +/- 0.01 vs 1.00 +/- 0.12, P < 0.01; protein: 0.06 +/- 0.05 vs 0.89 +/- 0.12, P < 0.01). Furthermore, levels of the tight junction markers occludin, ZO-1 and claudin decreased in ALF rats compared with healthy control rats (mRNA: 0.60 +/- 0.09 vs 1.00 +/- 0.12, 0.11 +/- 0.00 vs 1.00 +/- 0.12 and 0.60 +/- 0.01 vs 1.00 +/- 0.08, respectively, P < 0.01; protein: 0.05 +/- 0.01 vs 0.87 +/- 0.40, 0.09 +/- 0.05 vs 0.89 +/- 0.18 and 0.04 +/- 0.03 vs 0.95 +/- 0.21, respectively, P < 0.01). In ALF rats treated with QGHXR, E-cadherin levels increased (mRNA: QGHXR 0.67 +/- 0.04 vs ALF model 0.16 +/- 0.05, P < 0.01; protein: QGHXR 0.66 +/- 0.21 vs ALF model 0.09 +/- 0.05, P < 0.01), and vimentin and fibronectin levels decreased (mRNA: 6.57 +/- 1.05 vs 11.43 +/- 0.39 and 1.45 +/- 1.51 vs 9.91 +/- 0.34, respectively, P < 0.01; protein: 0.09 +/- 0.03 vs 1.13 +/- 0.42 and 0.10 +/- 0.01 vs 1.16 +/- 0.43, respectively, P < 0.01). In addition, QGHXR inhibited the expression of TGF-beta1 and increased the expression of Smad3 (mRNA: 1.03 +/- 0.11 vs 1.76 +/- 0.12, 0.70 +/- 0.10 vs 0.05 +/- 0.01, respectively, P < 0.05 and P < 0.01; protein: 0.12 +/- 0.03 vs 1.43 +/- 0.05 and 0.88 +/- 0.20 vs 0.06 +/- 0.05, respectively, P < 0.01). QGHXR treatment also reduced the levels of the EMT-inducing transcription factor snail (mRNA: 2.28 +/- 0.33 vs 6.98 +/- 0.41, P < 0.01; protein: 0.08 +/- 0.02 vs 1.07 +/- 0.29, P < 0.01) and increased the occludin, ZO-1 and claudin levels (mRNA: 0.73 +/- 0.05 vs 0.60 +/- 0.09, 0.57 +/- 0.04 vs 0.11 +/- 0.00 and 0.68 +/- 0.03 vs 0.60 +/- 0.01, respectively, P < 0.01, P < 0.01 and P < 0.05; protein: 0.92 +/- 0.50 vs 0.05 +/- 0.01, 0.94 +/- 0.22 vs 0.09 +/- 0.05 and 0.94 +/- 0.29 vs 0.04 +/- 0.03, respectively, P < 0.01). The effects of QGR and HXR on the TGF-beta1/Smad signaling pathway were similar to that of QGHXR; however, the QGR- and HXR-induced changes in vimentin mRNA levels, the QGR-induced changes in fibronectin mRNA levels and the HXR-induced changes in snail and TGF-beta1 mRNA levels were not significant. CONCLUSION: Qinggan Huoxue Recipe inhibits EMT in ALF rats by modulating the TGF-beta1/Smad signaling pathway, suggesting that the mechanism underlying the amelioration of ALF induced by QGHXR is associated with this pathway.

epithelial-mesenchymal transition (EMT) is a process associated with airway remodeling in chronic obstructive pulmonary disease (COPD), which leads to progressive pulmonary destruction. Panax ginseng is a traditional herbal medicine that has been shown to improve pulmonary function and exercise capacity in patients with COPD. Ginsenoside Rg1 is one of the main active components and was shown to inhibit oxidative stress and inflammation. The present study investigated the hypothesis that ginsenoside Rg1 attenuates EMT in COPD rats induced by cigarette smoke (CS) and human bronchial epithelial (HBE) cells exposed to cigarette smoke extract (CSE). Our data showed that CS or CSE exposure increased expression of the mesenchymal marker alpha-smooth muscle actin (alpha-SMA) and decreased expression of the epithelial marker epithelial cadherin (E-cad) in both lung tissues and HBE cells, which was markedly suppressed by ginsenoside Rg1. Importantly, CS-induced upregulation of TGF-beta1/Smad pathway components, including TGF-beta1, TGF-betaR1, phospho-Smad2, and phospho-Smad3, was also inhibited by ginsenoside Rg1. Additionally, ginsenoside Rg1 mimicked the effect of SB525334, a TGF-betaR1-Smad2/3 inhibitor, on suppression of EMT in CSE-induced HBE cells. Collectively, we concluded that ginsenoside Rg1 alleviates CS-induced pulmonary EMT, in both COPD rats and HBE cells, via inhibition of the TGF-beta1/Smad pathway.