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Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease that typically leads to respiratory failure and death within 3-5 years of diagnosis. Sub-pleural pulmonary fibrosis is a pathological hallmark of IPF. Bleomycin treatment of mice is a an established pulmonary fibrosis model. We recently showed that bleomycin-induced epithelial-mesenchymal transition (EMT) contributes to pleural mesothelial cell (PMC) migration and sub-pleural pulmonary fibrosis. MicroRNA (miRNA) expression has recently been implicated in the pathogenesis of IPF. However, changes in miRNA expression in PMCs and sub-pleural fibrosis have not been reported. Using cultured PMCs and a pulmonary fibrosis animal model, we found that miR-18a-5p was reduced in PMCs treated with bleomycin and that downregulation of miR-18a-5p contributed to EMT of PMCs. Furthermore, we determined that miR-18a-5p binds to the 3' UTR region of transforming growth factor β receptor II (TGF-βRII) mRNA, and this is associated with reduced TGF-βRII expression and suppression of TGF-β-Smad2/3 signaling. Overexpression of miR-18a-5p prevented bleomycin-induced EMT of PMC and inhibited bleomycin-induced sub-pleural fibrosis in mice. Taken together, our data indicate that downregulated miR-18a-5p mediates sub-pleural pulmonary fibrosis through upregulation of its target, TGF-βRII, and that overexpression of miR-18a-5p might therefore provide a novel approach to the treatment of IPF.

Interleukin 17 (IL-17) plays important roles in the progression of asthma. Genetic variants in the Il-17 may influence the immunopathogenesis of many diseases. Many studies have investigated the relevance of IL-17 polymorphism with cancers or immune diseases, including asthma. In this study, single nucleotide polymorphisms (SNPs) of IL-17 were explored by PCR-RFLP and verified by sequencing method. The frequencies of genotypes and alleles were analyzed. Haplotypes were analyzed with the SHEsis online program. The relationship between the genotypes of SNPs and IgE level was also investigated. The False Discovery Rate (FDR) correction was performed (P-adjusted 0.05). The frequencies of A allele, GA and (GA + AA) genotype of rs3748067 were significantly higher in asthma patients. As for rs763780, the C allele in patients was more frequent than healthy controls. In addition, we found C carriers (CT + CC) were significantly higher in asthma patients. We further found that the haplotype CT for IL-17F (rs763780/rs2397084) was associated with an increased susceptibility of asthma, but this association did not survive after FDR correction. The level of serum total IgE in mutant group (GA + AA) of rs3748067 was significantly higher than the wild genotype (GG) group and control group. These results suggested that IL-17 SNPs, but not haplotypes may be associated with the susceptibility of asthma in Chinese Han population from central China.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease of unknown cause that typically leads to respiratory failure and death within 3–5years of diagnosis. TGF-β1 is considered a major profibrotic factor. However, TGF-β1 is necessary but not sufficient to the pathogenesis of fibrotic lesion of the lungs. Recent observations have revealed that calpain, a calcium dependent protease, plays a pivotal role in tissue remodeling and fibrosis. However, the mechanism of calpain mediating pulmonary fibrosis is not understood. Calpain conditional knockout (ER-Cre+/−capns1flox/flox) mice and primary human lung fibroblasts (HLFs) were used here to investigate the relationship between calpain and TGF-β1. Calpain knockout mice were protected from fibrotic effects of bleomycin. Bleomycin induced increases in TGF-β1 via calpain activation in HLFs. Moreover, TGF-β1 also activated calpain. This crosstalk between calpain activation and TGF-β1 triggered the downstream signaling pathway including TGF-β1 Smad2/3 and non-Smad (Akt) pathways, as well as collagen-I synthesis. Taken together, our data indicate that the crosstalk between calpain activation and TGF-β1 augments collagen-I synthesis in HLFs and in pulmonary fibrosis. Intervention in the crosstalk between calpain activation and TGF-β1 is a novel potential strategy to prevent pulmonary fibrosis.

Background Mucus overproduction is an important feature of asthma. Interleukin (IL)-4 is required for allergen-induced airway inflammation and mucus production. MUC5AC gene expression is regulated by transcript factors NF-κB. The intracellular Ca2+ ([Ca2+]i) signal is required for activation of NF-κB. The transient receptor potential canonical 1 (TRPC1) channel has been shown to contribute for agonist-stimulated Ca2+ influx in some types of cells. However, the relationships among IL-4, TRPC1 and mucus overproduction in bronchial epithelial cells (BECs) in asthma are poorly understood. Methods BECs were isolated from large bronchial airway of rats and used as cell model. To present changes of lipid raft, caveolin-1 and TRPC1, immunofluorescence staining and sucrose gradient centrifugation were performed. [Ca2+]i was measured after loading with Fura-2. NF-κB activities were measured by an ELISA-based assay. MUC5AC mRNA and protein levels were detected by real-time quantitative RT-PCR, ELISA analysis and immunofluorescence staining respectively. Results IL-4 induced Ca2+ influx in BECs, and this was blocked by a Ca2+ influx inhibitor (2-APB). 2-APB also prevented MUC5AC protein synthesis induced by IL-4. Depletion of extracellular Ca2+ resulted in partial decrease in expression of MUC5AC in IL-4 treated cells. NF-κB rather than STAT6 activation mediated IL-4-induced MUC5AC protein synthesis. Then the mechanism of Ca2+ influx was investigated. Immunofluorescence staining and sucrose gradient centrifugation revealed that caveolin-1-containing lipid rafts aggregation was involved in TRPC1 activation and Ca2+ influx in BECs. Lastly, the data revealed that blocking lipid rafts aggregation exactly prevented Ca2+ influx, NF-κB activation and MUC5AC synthesis induced by IL-4. Conclusions Our results indicate that IL-4-induced caveolin-1-containing lipid rafts aggregation at least partly contributes to MUC5AC synthesis in BECs. Electronic supplementary material The online version of this article (10.1186/s12931-017-0657-z) contains supplementary material, which is available to authorized users.

Background Plasma miR-21 is widely investigated as biomarker in many diseases. Recent studies show that miR-21 participates in the development of systemic lupus erythematosus (SLE). The aim of this study was to evaluate the expression profile of miR-21 in the plasma of SLE patients. Methods Relative quantities of plasma miR-21 both in SLE patients and healthy controls were determined by relative qRT-PCR under endogenous and exogenous controls. The diagnostic value of plasma miR-21 was evaluated in SLE patients. Data of some SLE-associated clinical parameters were collected. Results Eighty participants from Central China were recruited. Forty-four participants were new-onset SLE patients and the others were healthy controls. Plasma miR-21 level in SLE patients was higher than that of healthy controls (P = 0.031). Receiver operating characteristic analysis of plasma miR-21 revealed an Area Under Curve (AUC) of 0.64 ± 0.06 (95% CI: 0.51–0.76, P = 0.03854) when differentiating SLE from healthy controls. The level of plasma miR-21 was not associated with the level of white blood cells (P = 0.4284), red blood cells (P = 0.4079), and platelets (P = 0.4961), but significantly correlated with the level of plasma complement C3 (r = −0.5297, P = 0.0004), C4 (r = −0.4732, P = 0.0020), and serum uric acid (r = 0.3932, P = 0.0121) in SLE patients. Conclusions Plasma miR-21 in SLE patients from Central China is overexpressed. Since circulating miR-21 is aberrantly expressed in many diseases, the applying of it as a disease biomarker should be considered carefully.

Radiation leads to a rapid burst of reactive oxygen species (ROS), which is considered to be one of the major causes of radiation-induced injury. ROS have previously been shown to induce changes in cytosolic Ca2+ ([Ca2+]i) including [Ca2+]i oscillation. However, the role of radiation in [Ca2+]i oscillation is poorly understood. The purpose of this study was to identify the effect of ROS and X ray on [Ca2+]i oscillation, as well as their role in radiation-induced lung injury. Alveolar macrophages were cultured in the absence and presence of different doses of hydrogen peroxide (H2O2) or exposed to X-ray irradiation with or without pretreatment of diphenyleneiodonium chloride (DPI, an inhibitor of NADPH oxidases) or tetrandrine (TET, a calcium entry blocker) and cytosolic Ca2+ concentration was detected by fluorescent Ca2+ indicator Fura-2. Rat radiation lung injury was induced in vivo by using 40 Gy X ray and DPI or TET was used to prevent radiation-induced lung injury. The results showed that there was sp...

Objectives This study aimed to investigate the association between polymorphisms of T-cell immunoglobulin and mucin domain molecule-3 (TIM-3) and Graves' disease (GD) in a Chinese population. Design and methods Genomic DNA was extracted from peripheral blood cells of the 182 GD patients and 150 control subjects. The TIM-3 gene polymorphic sites were genotyped. We also analyzed the relationships between the genotypes of each SNP and serum specific clinical variables. To detect whether the variants were associated with the TIM-3 expression, we further studied 40 patients by using the method of real-time quantitative reverse-transcription polymerase chain reaction (real-time RT-PCR). Results The genotype and allele frequency of each polymorphic site were not significantly different between GD and control individuals. Furthermore, it also showed no relationship between the variants and TIM-3 mRNA expression. Conclusions Our results demonstrated that the polymorphisms of TIM-3 gene may not contribute to GD susceptibility in the Chinese Han population.

Background Allergic asthma is characterized by inflammation and airway remodeling. Airway remodeling with excessive deposition of extracellular matrix (ECM) and larger smooth muscle mass are correlated with increased airway responsiveness and asthma severity. Calpain is a family of calcium-dependent endopeptidases, which plays an important role in ECM remodeling. However, the role of calpain in airway smooth muscle remodeling remains unknown. Objective To investigate the role of calpain in asthmatic airway remodeling as well as the underling mechanism. Methods The mouse asthma model was made by ovalbumin sensitization and challenge. Calpain conditional knockout mice were studied in the model. Airway smooth muscle cells (ASMCs) were isolated from smooth muscle bundles in airway of rats. Cytokines IL-4, IL-5, TNF-α and TGF-β1, and serum from asthmatic patients were selected to treated ASMCs. Collagen-I synthesis, cell proliferation, and phosphorylation of Akt in ASMCs were analyzed. Results Inhibition of calpain using calpain knockout mice attenuated airway smooth muscle remodeling in mouse asthma models. Cytokines IL-4, IL-5, TNF-α and TGF-β1, and serum from asthmatic patients increased collagen-I synthesis, cell proliferation, and phosphorylation of Akt in ASMCs, which were blocked by the calpain inhibitor MDL28170. Moreover, MDL28170 reduced cytokine-induced increases in Rictor protein which is the most important component of mammalian target of rapamycin complex 2 (mTORC2). Blockage of the mTORC2 signal pathway prevented cytokine-induced phosphorylation of Akt, collagen-I synthesis and cell proliferation of ASMCs, and attenuated airway smooth muscle remodeling in mouse asthma models. Conclusions Our results indicate that calpain mediates cytokine-induced collagen-I synthesis and proliferation of ASMCs via the mTORC2/Akt signaling pathway, thereby regulating airway smooth muscle remodeling in asthma. This article is protected by copyright. All rights reserved.

Pleural fibrosis is defined as an excessive deposition of extracellular matrix (ECM) components that results in destruction of the normal pleural tissue architecture. It can result from diverse inflammatory conditions, especially tuberculous pleurisy. Pleural mesothelial cells (PMCs) play a pivotal role in pleural fibrosis. Calpain is a family of calcium-dependent endopeptidases, which plays an important role in ECM remodeling. However, the role of calpain in pleural fibrosis remains unknown. In the present study, we found that tuberculous pleural effusion (TPE) induced calpain activation in PMCs and that inhibition of calpain prevented TPE-induced collagen-I synthesis and cell proliferation of PMCs. Moreover, our data revealed that the levels of angiotensin (ANG)-converting enzyme (ACE) were significantly higher in pleural fluid of patients with TPE than those with malignant pleural effusion, and ACE-ANG II in TPE resulted in activation of calpain and subsequent triggering of the phosphatidylinositol 3-kinase (PI3K)/Akt/NF-κB signaling pathway in PMCs. Finally, calpain activation in PMCs and collagen depositions were confirmed in pleural biopsy specimens from patients with tuberculous pleurisy. Together, these studies demonstrated that calpain is activated by renin-angiotensin system in pleural fibrosis and mediates TPE-induced collagen-I synthesis and proliferation of PMCs via the PI3K/Akt/NF-κB signaling pathway. Calpain in PMCs might be a novel target for intervention in tuberculous pleural fibrosis.

Exposed to inflammatory factors or cytokines, fibroblasts appear to play additional roles beyond the deposition of extracellular matrix. It has been reported that tumor necrosis factor-α (TNF-α) induces the production of matrix metalloproteinase-2 (MMP-2) and transforming growth factor-β1 (TGF-β1) in fibroblasts. In this study, we demonstrated that the active MMP-2 secreted by lung fibroblasts reached the peak level at 12 hours after TNF-α treatment, whereas, by adding anti-TGF-β1 antibody in the culture medium, the MMP-2 production in response to TNF-α was maintained at high levels after 24 hours of treatment. We also confirmed that TNF-α induced up-regulation of active TGF-β1 and exogenous TGF-β1 induced down-regulation of MMP-2 synthesis in lung fibroblasts. Moreover, an increased MMP-2 level was observed in a rat model with pulmonary inflammation and fibrosis induced by bleomycin-A5. This revealed that MMP-2 in the lung reached the peak level when TNF-α reached the peak level at the 7th day, and then MMP-2 decreased along with an increase in the TGF-β1 level. Taken together, our results demonstrate that TNF-α induced an increase of MMP-2 and TGF-β1 in lung fibroblasts, and the TGF-β1 attenuated the up-regulation of MMP-2. This suggests that MMP-2 secreted from fibroblasts modulated by TNF-α/TGF-β1 might play an important role in pulmonary inflammation and fibrosis.

Over 80 years ago, Warburg identified a particular metabolic pathway in carcinomas characterised by the anaerobic degradation of glucose even in the presence of oxygen that leads to the production of large amounts of lactate (known as the Warburg effect). Now, widespread clinical use of positron-emission tomography (PET) has confirmed that there exists enhanced glucose degradation in tumors. Recent research demonstrated that pentose phosphate pathway (PPP) was augmented in some tumors, especially non-oxidative part of PPP. The non-oxidative part of PPP is controlled by transketolase enzyme reactions. The present study designed to evaluate the effect of transketolase activity on nasopharyngeal carcinoma. It was found that the transketolase activity was significantly stronger in human nasopharyngeal carcinoma tissues than those in human chronic nasopharyngitis tissues. There is a strong upregulation of the transketolase-like-1 (TKTL1) in human nasopharyngeal carcinoma tissues and cell line (CNE), whereas transketolase (TKT) and transketolase-like-2 (TKTL2) were not upregulated. After inhibited the expression of (TKTL1) by RNAi, we found that total transketolase activity was dramatically downregulated and the proliferation of cancer cells was significantly inhibited in CNE cells. These results indicate that TKTL1 gene influences total transketolase activity and cell proliferation in human nasopharyngeal carcinoma cells, suggesting that TKTL1 gene plays an important role on glycometabolism in tumors and it might become a novel target for tumor gene therapy.

Airway hyperresponsiveness (AHR) in asthma is predominantly caused by increased sensitivity of bronchial smooth muscle cells (BSMCs) to stimuli. The sarcoplasmic reticulum (SR)-Ca(2+) release channel, known as ryanodine receptor (RyR), mediates the contractive response of BSMCs to stimuli. FK506-binding protein 12.6 kD (FKBP12.6) stabilizes the RyR2 channel in a closed state. However, the interaction of FKBP12.6 with RyR2 in AHR remains unknown. This study examined the interaction of FKBP12.6 with RyR2 in BSMCs in AHR of asthma. The interaction of FKBP12.6 with RyR2 and FKBP12.6 expression was determined in a rat asthma model and in BSMCs treated with inflammatory cytokines. The calcium responses to contractile agonists were determined in BSMCs with overexpression and knockdown of FKBP12.6. Asthmatic serum, IL-5, IL-13, and TNF-α enhance the calcium response of BSMCs to contractile agonists and cause dissociation of FKBP12.6 from RyR2 and a decrease in FKBP12.6 gene expression in BSMCs in culture and in ovalbumin (OVA)-sensitized and -challenged rats. Knockdown of FKBP12.6 in BSMCs causes a decrease in the association of RyR2 with FKBP12.6 and an increase in the calcium response of BSMCs. Overexpression of FKBP12.6 increases the association of FKBP12.6 with RyR2, decreases the calcium response of BSMCs, and normalizes airway responsiveness in OVA-sensitized and -challenged rats. Dissociation of FKBP12.6 from RyR2 in BSMCs is responsible for the increased calcium response contributing to AHR in asthma. Manipulating the interaction of FKBP12.6 with RyR2 might be a novel and useful treatment for asthma.

Radiation leads to a rapid burst of reactive oxygen species (ROS), which is considered to be one of the major causes of radiation-induced injury. ROS have previously been shown to induce changes in cytosolic Ca²⁺ ([Ca²⁺]i) including [Ca²⁺]i oscillation. However, the role of radiation in [Ca²⁺]i oscillation is poorly understood. The purpose of this study was to identify the effect of ROS and X ray on [Ca²⁺]i oscillation, as well as their role in radiation-induced lung injury. Alveolar macrophages were cultured in the absence and presence of different doses of hydrogen peroxide (H₂O₂) or exposed to X-ray irradiation with or without pretreatment of diphenyleneiodonium chloride (DPI, an inhibitor of NADPH oxidases) or tetrandrine (TET, a calcium entry blocker) and cytosolic Ca²⁺ concentration was detected by fluorescent Ca²⁺ indicator Fura-2. Rat radiation lung injury was induced in vivo by using 40 Gy X ray and DPI or TET was used to prevent radiation-induced lung injury. The results showed that there was spontaneous [Ca²⁺]i oscillation in alveolar macrophages under normal conditions, and treatment of H₂O₂ (100-500 μM) or 2 Gy X ray inhibited the spontaneous [Ca²⁺]i oscillation and induced [Ca²⁺]i rise. TET abolished H₂O₂ or X ray induced [Ca²⁺]i rise in alveolar macrophages, and attenuated X ray- induced rat alveolitis in vivo. DPI prevented X-ray-induced inhibition of [Ca²⁺]i oscillation in alveolar macrophages and prevented X-ray-induced rat alveolitis. Taken together, the data suggest that the disruption of [Ca²⁺]i oscillation and induction of [Ca²⁺]i rise through ROS is involved in the mechanism of radiation-induced lung injury.

Exposed to inflammatory factors or cytokines, fibroblasts appear to play additional roles beyond the deposition of extracellular matrix. It has been reported that tumor necrosis factor-α (TNF-α) induces the production of matrix metalloproteinase-2 (MMP-2) and transforming growth factor-β1 (TGF-β1) in fibroblasts. In this study, we demonstrated that the active MMP-2 secreted by lung fibroblasts reached the peak level at 12 hours after TNF-α treatment, whereas, by adding anti-TGF-β1 antibody in the culture medium, the MMP-2 production in response to TNF-α was maintained at high levels after 24 hours of treatment. We also confirmed that TNF-α induced up-regulation of active TGF-β1 and exogenous TGF-β1 induced down-regulation of MMP-2 synthesis in lung fibroblasts. Moreover, an increased MMP-2 level was observed in a rat model with pulmonary inflammation and fibrosis induced by bleomycin-A5. This revealed that MMP-2 in the lung reached the peak level when TNF-α reached the peak level at the 7th day, and then MMP-2 decreased along with an increase in the TGF-β1 level. Taken together, our results demonstrate that TNF-α induced an increase of MMP-2 and TGF-β1 in lung fibroblasts, and the TGF-β1 attenuated the up-regulation of MMP-2. This suggests that MMP-2 secreted from fibroblasts modulated by TNF-α/TGF-β1 might play an important role in pulmonary inflammation and fibrosis.

To investigate the effects of transketolase-like gene TKTL1 on the occurrence and metastasis of human nasopharyngeal carcinoma (NPC).Real-time PCR was used to determine the mRNA expression levels of transketolase gene family (TKT, TKTL1, and TKTL2) in the 65 biopsy specimens of human nasopharyngeal carcinoma, 9 at stage I, 15 at stage II, 16 at stage III, 13 at stage IVA, and 12 at stage IVB, 42 with metastasis and 232 without metastasis, and 9 biopsy specimens of chronic nasopharyngitis.The TKL activity level of the NPC tissues was 21.6 +/- 2.8, significantly higher than that of the chronic nasopharyngitis tissues (6.2 +/- 1.3, P0.01). The TKL activity level of the NPC tissues with metastasis was 26.5 +/- 3.2, significantly higher than that of the NPC tissues without metastasis (17.6 +/- 2.1, P0.01). There was no significant difference in the expression levels of TKT and TKTL2 genes between the human NPC and chronic nasopharyngitis tissues (both P0.05). The expression level of TKTL1 gene in the NPC tissues was 5.25 +/- 0.32, significantly higher than that in the chronic nasopharyngitis tissues (0.98 +/- 0.11, t = 6.23, P0.01), and the expression level of TKTL1 gene in the NPC tissues with lymph node metastasis was 5.86 +/- 0.38, significantly higher than that in the NPC tissues without lymph node metastasis (4.18 +/- 0.22, t = 3.28, P0.01).TKTL1 may play an important role in the occurrence and metastasis of human NPC and become a potential target for novel anti-cancer therapy.

A plasmid carrying DNA to be transcribed into a small interfering RNA against transketolase-like-1 mRNA was constructed and transfected into a human colon cancer cell line. The mRNA expression of transketolase gene family in the human colon cell line was determined by real-time polymerase chain reaction. The effect of anti-transketolase-like-1 small interfering RNA on cell proliferation and cell cycle in the human colon cancer cell line cells was detected by flow cytometry and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide. The transketolase-like-1 gene was significantly downregulated in human colon cancer cell line cells transfected with small interfering RNA transketolase-like-1 constructs compared with the cells transfected with control vector and the cells without transfection. In addition, the anti-transketolase-like-1 small interfering RNA construct significantly decreased the level of transketolase in the transfected human colon cancer cell line cells, arrested them in G0/G1 phase and substantially inhibited cell proliferation. No significant difference was found in the other two genes (transketolase and transketolase-like-2 genes) between the transfected human colon cancer cell line cells and the controls (P0.05). Our data demonstrated that the transketolase-like-1 gene plays an important role in total transketolase activity and in the cell proliferation of human colon cancer. Transketolase-like-1 may serve as a target for novel anticancer therapies.

We detected a strong upregulation of the mutated transketolase transcript (TKTL1) in human hepatoma cell line HepG2, whereas transketolase (TKT) and transketolase-like-2 (TKTL2) transcripts were not upregulated. We inhibited the expression of TKTL1 by RNAi in HepG2 cells. It was found that total transketolase activity was dramatically downregulated and the proliferation of cancer cells was significantly inhibited in HepG2 cells. These results indicate that TKTL1 gene influences total transketolase activity and cell proliferation in human hepatoma cells, suggesting that TKTL1 gene plays an important role on glycometabolism in tumors and it might become a novel target for tumor gene therapy.

This article has been retracted at the request of the Editor-in-Chief. Retraction: Overexpression of transketolase protein TKTL1 is associated with occurrence and progression in nasopharyngeal carcinoma: A potential therapeutic target in nasopharyngeal carcinoma Song Zhang, Jian-Xin Yue, Ju-Hong Yang, Peng-Cheng Cai and Wei-Jia Kong Reason: In the process of investigating an accusation of misconduct in the above manuscript, the Editors have found significant evidence of fraud perpetrated by some of the authors.1) Dr. Wei Jia Kong's name was used as the corresponding author without his knowledge or consent. Furthermore, Dr. Cai Pengcheng was unaware of his status as an author of the manuscript.2) A fake email address for Dr. Kong was constructed and used by the authors to intercept any information that would be sent to the corresponding author.3) Dr. Zhang Song and Ms. Yang Juhong have modified their accounts of the events several times during the investigation, making it difficult to determine exactly wha...

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease characterized by the development of subpleural foci of myofibroblasts that contribute to the exuberant fibrosis. Recent studies revealed that pleural mesothelial cells (PMCs) undergo epithelial–mesenchymal transition (EMT) and play a pivotal role in IPF. In animal model, bleomycin induces pulmonary fibrosis exhibiting subpleural fibrosis similar to what is seen in human IPF. It is not known yet whether bleomycin induces EMT in PMCs. In the present study, PMCs were cultured and treated with bleomycin. The protein levels of collagen-I, mesenchymal phenotypic markers (vimentin and α-smooth muscle actin), and epithelial phenotypic markers (cytokeratin-8 and E-cadherin) were measured by Western blot. PMC migration was evaluated using wound-healing assay of culture PMCs in vitro, and in vivo by monitoring the localization of PMC marker, calretinin, in the lung sections of bleomycin-induced lung fibrosis. The results showed that bleomycin induced increases in collagen-I synthesis in PMC. Bleomycin induced significant increases in mesenchymal phenotypic markers and decreases in epithelial phenotypic markers in PMC, and promoted PMC migration in vitro and in vivo. Moreover, TGF-β1-Smad2/3 signaling pathway involved in the EMT of PMC was demonstrated. Taken together, our results indicate that bleomycin induces characteristic changes of EMT in PMC and the latter contributes to subpleural fibrosis.