Expression In Lung Fibroblasts Research Articles

Suberanilohydroxamic acid prevents TGF-β1-induced COX-2 repression in human lung fibroblasts post-transcriptionally by TIA-1 downregulation

Cyclooxygenase-2 (COX-2), with its main antifibrotic metabolite PGE2, is regarded as an antifibrotic gene. Repressed COX-2 expression and deficient PGE2 have been shown to contribute to the activation of lung fibroblasts and excessive deposition of collagen in pulmonary fibrosis. We have previously demonstrated that COX-2 expression in lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) is epigenetically silenced and can be restored by epigenetic inhibitors. This study aimed to investigate whether COX-2 downregulation induced by the profibrotic cytokine transforming growth factor-β1 (TGF-β1) in normal lung fibroblasts could be prevented by epigenetic inhibitors. We found that COX-2 protein expression and PGE2 production were markedly reduced by TGF-β1 and this was prevented by the pan-histone deacetylase inhibitor suberanilohydroxamic acid (SAHA) and to a lesser extent by the DNA demethylating agent Decitabine (DAC), but not by the G9a histone methyltransferase (HMT) inhibitor BIX01294 or the EZH2 HMT inhibitor 3-deazaneplanocin A (DZNep). However, chromatin immunoprecipitation assay revealed that the effect of SAHA was unlikely mediated by histone modifications. Instead 3′-untranslated region (3′-UTR) luciferase reporter assay indicated the involvement of post-transcriptional mechanisms. This was supported by the downregulation by SAHA of the 3′-UTR mRNA binding protein TIA-1 (T-cell intracellular antigen-1), a negative regulator of COX-2 translation. Furthermore, TIA-1 knockdown by siRNA mimicked the effect of SAHA on COX-2 expression. These findings suggest SAHA can prevent TGF-β1-induced COX-2 repression in lung fibroblasts post-transcriptionally through a novel TIA-1-dependent mechanism and provide new insights into the mechanisms underlying its potential antifibrotic activity. AbbreviationsUnlabelled TableSAHAsuberanilohydroxamic acidTGF-β1transforming growth factor-β1COX-2cyclooxygenase-2TIA-1T-cell intracellular antigen-1PGE2prostaglandin E2IPFidiopathic pulmonary fibrosisDACDecitabineHMThistone methyltransferaseEZH2enhancer of zeste homolog 2DZNep3-deazaneplanocin A3′-UTR3′-untranslated regionα-SMAα-smooth muscle actinECMextracellular matrixCOL1collagen 1DNMTDNA methyltransferaseHAThistone acetyltransferaseHDAChistone deacetylaseH3K9me3histone H3 lysine 9 trimethylationAREAUUUA-rich elementHuRhuman antigen RELAV1ELAV-like RNA binding protein 1TTPTristetraprolinCUGBP2CUG triplet repeat, RNA binding protein 2F-NLfibroblast from non-fibrotic lungFCSfetal calf serum

Therapeutic administration of inhaled INS1009, a treprostinil prodrug formulation, inhibits bleomycin-induced pulmonary fibrosis in rats

Idiopathic pulmonary fibrosis is a progressive and lethal disease and while there are now two approved drugs (Esbriet® and Ofev®) additional effective treatments are still needed. Recently, prostacyclin analogs such as iloprost and treprostinil (TRE) have been shown to exert some protection against bleomycin-induced pulmonary fibrosis in mice when administered in a prophylactic regimen. In this study, we evaluated the effect of the inhaled treprostinil prodrug hexadecyl-treprostinil (C16TR) formulated in a lipid nanoparticle (INS1009) administered therapeutically in a fibrotic rat model. Male Fischer 344 rats challenged with intra-tracheal saline instillation were then treated with daily inhaled phosphate buffered saline (PBS) while rats challenged with bleomycin sulfate (3.5–4.0 mg/kg) instillation were treated with either daily inhaled PBS, daily inhaled INS1009 (10, 30, or 100 μg/kg), or twice-daily orally with the anti-fibrotic compound pirfenidone (100 mg/kg). Dosing started on day 10 post-bleomycin challenge and continued until day 27 after bleomycin. Lungs were harvested 24 h after the last dose of treatment for evaluation of lung hydroxyproline content and pulmonary histology. Lung hydroxyproline content increased from 421 μg/lung lobe in saline challenged and PBS treated animals to 673 μg/lung lobe in bleomycin challenged and PBS treated rats. Treatment of bleomycin challenged rats with 10, 30, or 100 μg/kg INS1009 dose-dependently reduced lung hydroxyproline content to 563, 501, and 451 μg/lung lobe, respectively, and pirfenidone decreased hydroxyproline content to 522 μg/lung lobe. Histologically, both INS1009 (100 μg/kg) and pirfenidone (100 mg/kg) reduced the severity of subepithelial fibrosis. Single dose pharmacokinetic (PK) studies of inhaled INS1009 in bleomycin challenged rats showed dose-dependent increases in lung C16TR concentration and plasma TRE on day 10 post-bleomycin challenge. Multiple dose PK studies of inhaled INS1009 showed dose-dependent increases only in lung C16TR concentration on day 27 post-bleomycin challenge. We also investigated the effects of TRE on the cytokine transforming growth factor-β1 (TGF-β1)-stimulated collagen gene and protein expressions in cultured human lung fibroblasts, assessed by real-time PCR and Sirius Red staining, respectively. In human fibroblasts, TRE (0.001–10 μM) inhibited TGF-β1 (20 ng/mL)-induced expression of collagen mRNA and protein in a concentration-dependent manner. These results demonstrated that inhaled INS1009, administered in a therapeutic dosing paradigm, dose-dependently (10–100 μg/kg) inhibited bleomycin-induced pulmonary fibrosis in rats. This effect may involve direct actions of TRE in suppressing collagen expression in lung fibroblasts.

Roles of high-mobility group box 1 and thrombin in murine pulmonary fibrosis and the therapeutic potential of thrombomodulin.

Cross talk between inflammation and coagulation plays important roles in acute or subacute progressive pulmonary fibrosis characterized by diffuse alveolar damage. Thrombomodulin is a physiological inhibitor of high-mobility group box 1 (HMGB1), and thrombin and may be effective for this condition. This study investigated the roles of HMGB1 and thrombin in the pathophysiology of bleomycin-induced pulmonary fibrosis and the efficacy of recombinant human soluble thrombomodulin (rhTM). Pulmonary fibrosis was induced in wild-type C57BL/6 mice by intratracheal instillation of bleomycin. We first assessed HMGB1, thrombin, transforming growth factor (TGF)-β1, and α-smooth muscle actin (SMA) levels in bronchoalveolar lavage fluid and lung tissue sections over time. Expression of HMGB1 and thrombin was elevated before that of TGF-β1 and α-SMA and remained high during the fibrotic phase after bleomycin instillation. We next examined whether in vitro stimulation with HMGB1 and thrombin induced expression of TGF-β1 and α-SMA in cultured alveolar macrophages and lung fibroblasts, respectively, by performing quantitative PCR, enzyme-linked immunosorbent assay, Western blot, and immunofluorescence analyses. HMGB1 and thrombin stimulation induced TGF-β1 production by alveolar macrophages, and thrombin stimulation also induced α-SMA expression in lung fibroblasts. Finally, we evaluated the effect of rhTM on bleomycin-induced pulmonary fibrosis. Compared with the vehicle control, both early and late-phase administration of rhTM suppressed the fibrotic process. Our results suggest that HMGB1 and thrombin were involved in the pathophysiology of pulmonary fibrosis via production of profibrotic proteins and that rhTM attenuated bleomycin-induced pulmonary fibrosis. rhTM may be a therapeutic option for acute or subacute pulmonary fibrosis.

TBX4 is involved in the super-enhancer-driven transcriptional programs underlying features specific to lung fibroblasts

Lung fibroblasts participate in the pathogenesis of respiratory diseases, including lung cancer and pulmonary fibrosis. Although fibroblasts are ubiquitous constituents of various organs, their cellular diversity among different organs has been poorly characterized. Here, we aimed to investigate the distinct gene signature of lung fibroblasts that represents its pulmonary origin and the underlying gene regulatory networks. Promoter-level differential expression analysis by cap analysis of gene expression (CAGE) sequencing revealed distinct gene expression patterns of fibroblasts derived from different anatomical sites and identified 88 coding genes with higher expression in lung fibroblasts relative to other fibroblasts. Multiple key transcription factors important for lung mesenchyme development, including the T-box transcription factors TBX2, TBX4, and TBX5 were enriched in this lung-specific signature and were associated with super-enhancers. TBX4 showed highly specific expression in lung fibroblasts and was required for cell proliferation and collagen gel contraction capacity. Transcriptome analysis revealed that TBX4 could broadly regulate fibroblast-related pathways and partly contribute to super-enhancer-mediated transcriptional programs. Of pathological importance, lung fibroblast-specific genes were globally downregulated in lung cancer-associated fibroblasts (CAFs). Notably, TBX2, TBX4, and TBX5 were downregulated and hypermethylated in lung CAFs, suggesting an association between epigenetic silencing of these factors and phenotypic alteration of lung fibroblasts in cancer. Our study highlights the importance of T-box transcription factors, especially TBX4, and super-enhancers in the roles of lung fibroblasts in pulmonary physiology and pathogenesis.

Identification of transforming growth factor-beta-regulated microRNAs and the microRNA-targetomes in primary lung fibroblasts

BackgroundLung fibroblasts are involved in extracellular matrix homeostasis, which is mainly regulated by transforming growth factor-beta (TGF-β), and are therefore crucial in lung tissue repair and remodeling. Abnormal repair and remodeling has been observed in lung diseases like COPD. As miRNA levels can be influenced by TGF-β, we hypothesized that TGF-β influences miRNA expression in lung fibroblasts, thereby affecting their function.Materials and methodsWe investigated TGF-β1-induced miRNA expression changes in 9 control primary parenchymal lung fibroblasts using miRNA arrays. TGF-β1-induced miRNA expression changes were validated and replicated in an independent set of lung fibroblasts composted of 10 controls and 15 COPD patients using qRT-PCR. Ago2-immunoprecipitation followed by mRNA expression profiling was used to identify the miRNA-targetomes of unstimulated and TGF-β1-stimulated primary lung fibroblasts (n = 2). The genes affected by TGF-β1-modulated miRNAs were identified by comparing the miRNA targetomes of unstimulated and TGF-β1-stimulated fibroblasts.ResultsTwenty-nine miRNAs were significantly differentially expressed after TGF-β1 stimulation (FDR<0.05). The TGF-β1-induced miR-455-3p and miR-21-3p expression changes were validated and replicated, with in addition, lower miR-455-3p levels in COPD (p<0.05). We identified 964 and 945 genes in the miRNA-targetomes of unstimulated and TGF-β1-stimulated lung fibroblasts, respectively. The TGF-β and Wnt pathways were significantly enriched among the Ago2-IP enriched and predicted targets of miR-455-3p and miR-21-3p. The miR-455-3p target genes HN1, NGF, STRADB, DLD and ANO3 and the miR-21-3p target genes HHEX, CHORDC1 and ZBTB49 were consistently more enriched after TGF-β1 stimulation.ConclusionTwo miRNAs, miR-455-3p and miR-21-3p, were induced by TGF-β1 in lung fibroblasts. The significant Ago2-IP enrichment of targets of these miRNAs related to the TGF-β and/or Wnt pathways (NGF, DLD, HHEX) in TGF-β1-stimulated fibroblasts suggest a role for these miRNAs in lung diseases by affecting lung fibroblast function.

Transforming growth factor β1 (TGFβ1) regulates CD44V6 expression and activity through extracellular signal-regulated kinase (ERK)-induced EGR1 in pulmonary fibrogenic fibroblasts

The appearance of myofibroblasts is generally thought to be the underlying cause of the fibrotic changes that underlie idiopathic pulmonary fibrosis. However, the cellular/molecular mechanisms that account for the fibroblast-myofibroblast differentiation/activation in idiopathic pulmonary fibrosis remain poorly understood. We investigated the functional role of hyaluronan receptor CD44V6 (CD44 containing variable exon 6 (v6)) for differentiation of lung fibroblast to myofibroblast phenotype. Increased hyaluronan synthesis and CD44 expression have been detected in numerous fibrotic organs. Previously, we found that the TGFβ1/CD44V6 pathway is important in lung myofibroblast collagen-1 and α-smooth-muscle actin synthesis. Because increased EGR1 (early growth response-1) expression has been shown to appear very early and nearly coincident with the expression of CD44V6 found after TGFβ1 treatment, we investigated the mechanism(s) of regulation of CD44V6 expression in lung fibroblasts by TGFβ1. TGFβ1-mediated CD44V6 up-regulation was initiated through EGR1 via ERK-regulated transcriptional activation. We showed that TGFβ1-induced CD44V6 expression is through EGR1-mediated AP-1 (activator protein-1) activity and that the EGR1- and AP-1-binding sites in the CD44v6 promoter account for its responsiveness to TGFβ1 in lung fibroblasts. We also identified a positive-feedback loop in which ERK/EGR1 signaling promotes CD44V6 splicing and found that CD44V6 then sustains ERK signaling, which is important for AP-1 activity in lung fibroblasts. Furthermore, we identified that HAS2-produced hyaluronan is required for CD44V6 and TGFβRI co-localization and subsequent CD44V6/ERK1/EGR1 signaling. These results demonstrate a novel positive-feedback loop that links the myofibroblast phenotype to TGFβ1-stimulated CD44V6/ERK/EGR1 signaling.

Increased expression of latent TGF-β-binding protein 4 affects the fibrotic process in scleroderma by TGF-β/SMAD signaling

Scleroderma is a fibrosis-related disorder characterized by cutaneous and internal organ fibrosis, and excessive collagen deposition in extracellular matrix (ECM) is a major cause of fibrosis. Transforming growth factor-β (TGF-β)/SMAD signaling has a central role in the pathogenesis of fibrosis by inducing abnormal collagen accumulation in ECM, and latent TGF-β-binding protein 4 (LTBP-4) affects the secretion of latent TGF-β to ECM. A previous study indicated that bleomycin (BLM) treatment increased LTBP-4 expression in lung fibroblasts of Thy-1 knockout mice with lung fibrosis, and LTBP-4 further promoted TGF-β bioavailability as well as SMAD3 phosphorylation. However, the expression and function of LTBP-4 in human scleroderma remain unclear. We aimed to investigate the potential role of LTBP-4 in scleroderma through clinical, in vivo and in vitro studies. LTBP-4 and TGF-β expressions were significantly upregulated in systemic scleroderma (SSc) patients' plasma compared with normal controls (LTBP-4, 1,215±100.2 vs 542.8±41.7 ng/ml, P<0.0001; TGF-β, 1.5±0.2 vs 0.7±0.1 ng/ml, P=0.0031), while no significant difference was found between localized scleroderma (LSc) and normal controls. The plasma concentrations of LTBP-4 and TGF-β were even higher in SSc patients with lung fibrosis (LTBP-4, 1462± 137.3 vs 892.8±113.4 ng/ml, P=0.0037; TGF-β, 2.0±0.4 vs 0.9±0.2 ng/ml, P=0.0212) and esophagus involvement (1390±134.4 vs 940.7±127.0 ng/ml, P=0.0269; TGF-β, 1.9±0.3 vs 0.9±0.2 ng/ml, P=0.0426). The area under receiver operating characteristics (ROC) curve of LTBP-4 was 0.86. Immunohistochemistry measurement also demonstrated a higher LTBP-4 expression in sclerotic skin tissue of LSc and SSc compared with normal controls. More positive fibroblasts were also found in BLM-induced scleroderma mouse model than the saline-treated group. In in vitro studies, knockdown of LTBP-4 in SSc skin fibroblasts prominently reduced downstream COL1A1, COL1A2, and COL3A1 mRNA level by 84%, 82%, and 43%, respectively, and other fibrosis-related genes' expression were also decreased. Furthermore, extracellular TGF-β level and the SMAD2/3 phosphorylation were inhibited through LTBP-4 knockdown treatment, suggesting that the knockdown of LTBP-4 reduced the collagen expression through TGF-β/SMAD signaling pathway. Taken together, these data suggest that LTBP-4 affects fibrotic process in scleroderma, and the high expression of LTBP-4 in SSc plasma may serve as a clinical biomarker in diagnosing this disease. In addition, this study also lays the theoretical foundation for targeting LTBP-4 as treatment of scleroderma.

HMGB1 induces lung fibroblast to myofibroblast differentiation through NF‑κB‑mediated TGF‑β1 release.

The proinflammatory factor high-mobility group box protein 1 (HMGB1) has been implicated in the pathogenesis of lung fibrosis; however, the role of HMGB1 in lung fibrosis remains unclear. It has previously been reported that nuclear factor (NF)-κB and transforming growth factor (TGF)-β1 may be involved in lung fibrosis. Therefore, the present study aimed to examine the potential molecular mechanisms that underlie HMGB1-induced lung fibrosis via the regulation of NF-κB and TGF-β1. The results demonstrated that HMGB1 stimulation increased the activation of NF-κB and the release of TGF-β1, as well as the expression of α-smooth muscle actin (α-SMA) and collagen I in human lung fibroblasts in vitro. In addition, inhibition of NF-κB activation blocked HMGB1-induced TGF-β1 release, as well as α-SMA and collagen I expression in lung fibroblasts. Preventing the release of TGF-β1 inhibited HMGB1-induced α-SMA and collagen I expression; however, it had no effect on NF-κB activation. Collectively, these findings indicate that HMGB1 induces fibroblast to myofibroblast differentiation of lung fibroblasts via NF-κB-mediated TGF-β1 release.

Epigenetic Regulation of Caveolin-1 Gene Expression in Lung Fibroblasts.

Fibrotic disorders are associated with tissue accumulation of fibroblasts. We recently showed that caveolin (Cav)-1 gene suppression by a profibrotic cytokine, transforming growth factor (TGF)-β1, contributes to fibroblast proliferation and apoptosis resistance. Cav-1 has been shown to be constitutively suppressed in idiopathic pulmonary fibrosis (IPF), but mechanisms for this suppression are incompletely understood. We hypothesized that epigenetic processes contribute to Cav-1 down-regulation in IPF lung fibroblasts, and after fibrogenic stimuli. Cav-1 expression levels, DNA methylation status, and histone modifications associated with the Cav-1 promoter were examined by PCR, Western blots, pyrosequencing, or chromatin immunoprecipitation assays in IPF lung fibroblasts, normal fibroblasts after TGF-β1 stimulation, or in murine lung fibroblasts after bleomycin injury. Methylation-specific PCR demonstrated methylated and unmethylated Cav-1 DNA copies in all groups. Despite significant changes in Cav-1 expression, no changes in DNA methylation were observed in CpG islands or CpG island shores of the Cav-1 promoter by pyrosequencing of lung fibroblasts from IPF lungs, in response to TGF-β1, or after bleomycin-induced murine lung injury, when compared with respective controls. In contrast, the association of Cav-1 promoter with the active histone modification mark, H3 lysine 4 trimethylation, correlated with Cav-1 down-regulation in activated/fibrotic lung fibroblasts. Our data indicate that Cav-1 gene silencing in lung fibroblasts is actively regulated by epigenetic mechanisms that involve histone modifications, in particular H3 lysine 4 trimethylation, whereas DNA methylation does not appear to be a primary mechanism. These findings support therapeutic strategies that target histone modifications to restore Cav-1 expression in fibroblasts participating in pathogenic tissue remodeling.

Epigenetic regulation of cyclooxygenase-2 by methylation of c8orf4 in pulmonary fibrosis.

Fibroblasts derived from the lungs of patients with idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc) produce low levels of prostaglandin (PG) E2, due to a limited capacity to up-regulate cyclooxygenase-2 (COX-2). This deficiency contributes functionally to the fibroproliferative state, however the mechanisms responsible are incompletely understood. In the present study, we examined whether the reduced level of COX-2 mRNA expression observed in fibrotic lung fibroblasts is regulated epigenetically. The DNA methylation inhibitor, 5-aza-2'-deoxycytidine (5AZA) restored COX-2 mRNA expression by fibrotic lung fibroblasts dose dependently. Functionally, this resulted in normalization of fibroblast phenotype in terms of PGE2 production, collagen mRNA expression and sensitivity to apoptosis. COX-2 methylation assessed by bisulfite sequencing and methylation microarrays was not different in fibrotic fibroblasts compared with controls. However, further analysis of the methylation array data identified a transcriptional regulator, chromosome 8 open reading frame 4 (thyroid cancer protein 1, TC-1) (c8orf4), which is hypermethylated and down-regulated in fibrotic fibroblasts compared with controls. siRNA knockdown of c8orf4in control fibroblasts down-regulated COX-2 and PGE2 production generating a phenotype similar to that observed in fibrotic lung fibroblasts. Chromatin immunoprecipitation demonstrated that c8orf4 regulates COX-2 expression in lung fibroblasts through binding of the proximal promoter. We conclude that the decreased capacity of fibrotic lung fibroblasts to up-regulate COX-2 expression and COX-2-derived PGE2 synthesis is due to an indirect epigenetic mechanism involving hypermethylation of the transcriptional regulator, c8orf4.

IL-27 enhances innate immunity of human pulmonary fibroblasts and epithelial cells through upregulation of TLR4 expression.

Lung tissue cells play an active role in the pathogenesis of pulmonary inflammatory diseases by releasing a variety of cytokines and chemokines. However, how lung tissue cells respond to microbial stimuli during pulmonary infections remains unclear. In this study, we found that patients with community-acquired pneumonia displayed increased IL-27 levels in bronchoalveolar lavage fluid and serum. We subsequently examined the immunopathological mechanisms for the activation of primary human lung fibroblasts and bronchial epithelial cells by IL-27. We demonstrated that IL-27 priming enhanced LPS-induced production of IL-6 and IL-8 from lung fibroblasts and bronchial epithelia cells via upregulating Toll-like receptor-4 (TLR4) expression. IL-27 upregulated TLR4 expression in lung fibroblasts through activation of Janus-activated kinase (JAK) and Jun NH2-terminal kinase (JNK) signaling pathways, and inhibition of the JAK pathway could partially decrease IL-27-induced TLR4 expression, while inhibition of JNK pathway could completely suppress IL-27-induced TLR4 expression. Our data suggest that IL-27 modulates innate immunity of lung tissue cells through upregulating TLR4 expression during pulmonary infections.

Interaction of long noncoding RNAs and microRNAs in the pathogenesis of idiopathic pulmonary fibrosis.

Long noncoding RNAs (lncRNAs) are transcribed RNAs with more than 200 nucleotides in length. A growing body of evidence supports the notion that lncRNAs act as competitive endogenous RNAs for microRNAs and play roles in physiological and pathological processes. Several studies have demonstrated the roles of microRNAs in the pathogenesis of idiopathic pulmonary fibrosis (IPF). However, it is unknown whether lncRNAs are involved in IPF. To investigate the roles of lncRNAs in IPF, we determined the interaction of lncRNAs and microRNAs by motif search and manual comparison. The sequences of the dysregulated microRNAs in IPF including miR-21, miR-31, miR-101, miR-29, miR-199, and let-7d were used to search NONCODE database containing 33,829 human lncRNAs. A total of 34 lncRNAs with potential binding sites to these microRNAs were identified. We then examined the expression levels of the identified lncRNAs in the lungs of IPF patients by real-time PCR. Of 34 lncRNAs, nine lncRNAs were dysregulated in the IPF lungs. Four of them were inversely correlated to the microRNA expression in IPF. Further studies revealed that silencing the lncRNA CD99 molecule pseudogene 1 (CD99P1) inhibited proliferation and α-smooth muscle actin expression of lung fibroblasts, while knockdown of the lncRNA n341773 increased collagen expression in lung fibroblasts. These results suggest that CD99P1 and n341773 may be involved in the regulation of lung fibroblast proliferation and differentiation. The identification of regulatory functions of lncRNAs in lung fibroblasts may provide new research directions for the therapy of IPF.

Inhibition of Micro‐RNA 146a Expression in Lung Fibroblasts by Fluticasone Propionate, Salmeterol Xinafoate, and Related Agents

Micro‐RNA (miR) 146a is induced by inflammatory stimuli, and its pro‐inflammatory targets suggest that it may limit the inflammatory response. Fluticasone propionate (FP) and salmeterol xinafoate (SX) are used together to treat asthma, an inflammatory disease. Their effects on interleukin 1‐beta (IL‐1β)‐induced expression of miR‐146a by HFL‐1 lung fibroblasts were evaluated by quantitative RT‐PCR. Exposure of cells to the anti‐inflammatory steroid FP (1 nM) for 24 hr caused a nearly 70% inhibition. Two beta‐2 adrenergic receptor agonists, the long‐acting SX (10 nM) and the short‐acting albuterol (Alb, 10 μM) also inhibited miR‐146a expression, but only by 25‐35%. Prostaglandin E2 (100 nM) and the direct adenylyl cyclase activator forskolin (Fsk, 30 μM) both inhibited miR‐146a expression similarly to SX and Alb, suggesting cAMP elevation as the mechanism for these agents. Two primary isolates of human lung fibroblasts showed similar decreases in miR‐146a as for HFL‐1 cells when treated with FP, SX, or Fsk. FP has anti‐inflammatory effects in the airways that are augmented by concurrent SX, yet both FP and SX reduced fibroblast expression of anti‐inflammatory miR‐146a. Whether this is a direct effect of these agents or due to their overall inhibition of inflammation remains to be defined. Reduction in miR‐146a levels by these agents might limit their anti‐inflammatory actions, perhaps to prevent excess inhibition of inflammatory pathways.Supported by Glaxo Smith Kline Respiratory Division Investigator‐Initiated Grant 200164.

Matrix metalloproteinase‐12 (MMP12) inhibits myofibroblast differentiation and lung fibrosis

Matrix metalloproteinase 12 (MMP12) is implicated in pathogenesis of emphysema; however its role in lung fibrosis is controversial. C/EBPβ represses MMP12 expression in lung fibroblasts, while C/EBPβ deficiency attenuates bleomycin (BLM) induced lung fibrosis and myofibroblast (MF) differentiation accompanied with de‐repression of MMP12 expression. In this study, the hypothesis that MMP12 inhibits MF differentiation and accompanying fibrosis was evaluated. Promoter analysis, gel shift assay and real time PCR analysis confirmed that C/EBPβ was a direct repressor of MMP12 gene expression. C/EBPβ deficiency induced MMP12 expression in fibroblasts, which was accompanied by diminished α‐smooth muscle actin (α‐SMA) expression. Moreover ectopic over‐expression of MMP12 suppressed α‐SMA expression. In contrast, MMP12 deficiency or treatment with an MMP12 inhibitor enhanced α‐SMA expression, thus confirming its suppressor role in MF differentiation. A potential mechanism was suggested by the ability of MMP12 to cleave N‐cadherin, which was inhibited by an MMP12 inhibitor; while over‐expression of N‐cadherin stimulated α‐SMA gene expression. Furthermore MMP12 deficient mice exhibited significantly enhanced BLM‐induced pulmonary fibrosis relative to that in wild type mice as evaluated by analysis of collagen deposition, MF differentiation and histopathology. These findings suggest that fibroblast MMP12 is an endogenous repressor of MF differentiation, perhaps by digestion of N‐cadherin, thus playing an important homeostatic role to maintain fibroblasts in an undifferentiated state and keeping fibrosis in check. This work was supported by National Institute of Health grants (HL052285, HL091775, HL112880 and DK020572).

Leptin Modulates Airway Remodeling Processes and Responses to Interleukin-13 in Lung Fibroblasts in a Murine Model of Chronic Allergic Airways Disease

L3 Leptin Modulates Airway Remodeling Processes and Responses to Interleukin-13 in Lung Fibroblasts in a Murine Model of Chronic Allergic Airways Disease Jennifer L. Ingram, Robert W. Sigmon, Barbara Theriot, Michael Ghio, Akhil Hegde, Dave Francisco, Julia K. L. Walker, Monica Kraft, MD; Duke University, Durham, NC. RATIONALE: Obese asthma is characterized by late-onset disease and reduced T-helper 2 (TH2)-driven inflammation. Leptin is an adipokine produced in elevated levels in obese individuals. Interleukin-13 (IL-13) is a TH2 cytokine that directs airway remodeling in asthma, including subepithelial fibrosis. We hypothesized that leptin acts to suppress lung fibroblast responses to IL-13 in obese asthma. METHODS: Wildtype (C57BL6), leptin-resistant (db/db), and leptindeficient (ob/ob) mice (n53 per group) were intranasally administered house dust mite (HDM) allergen (2.5 mg/mL) or saline as control 5 days/ week for 6 weeks. Lung mechanics were measured using the forced oscillation technique, and Newtonian resistance was calculated. Lung sections were stained with Masson’s trichrome. Lung fibroblasts were cultured from digested lung tissue and exposed to serum-free media or 50 ng/mL IL-13 for 24 hours. Lung fibroblast invasion and migration were determined using Matrigel assays, and mRNA expression was analyzed using quantitative RT-PCR. RESULTS: Following exposure to HDM, peribronchiolar trichrome staining and IL-13-induced lung fibroblast invasion and migration were significantly reduced in ob/ob and db/db mice as compared to wildtype (p<0.05). IL-13-stimulated elastin gene expression in lung fibroblasts was significantly increased in ob/ob mice as compared to wildtype (p<0.05). Compared to saline treatment, exposure to HDM resulted in significantly increased Newtonian airway resistance (p<0.05) in wildtype and db/db mice, but not ob/ob mice. Saline-treated ob/ob mice were hyperresponsive and displayed a paradoxical response to methacholine challenge. CONCLUSIONS: Lung fibroblasts from leptin-deficient and –resistant mice demonstrate impaired responses to IL-13, which may contribute to altered airway remodeling and lung mechanics in obese asthma.

Inhibition of transforming growth factor-β via the activin receptor-like kinase-5 inhibitor attenuates pulmonary fibrosis.

Idiopathic pulmonary fibrosis is a chronic pulmonary disease that is characterized by formation of scar tissue in lungs. Transforming growth factor-β (TGF-β) is considered an important cytokine in the pathogenesis of this disease. Hence, the antifibrotic effect of an inhibitor of the TGF-β type I receptor, namely, SB 431542, was investigated in our study. SB 431542 was used to treat TGF-β-treated IMR-90 cells; the expression of α-smooth muscle actin (α-SMA) was detected at the protein level by using an anti-α-SMA antibody, and at the gene level by reverse transcription-quantitative PCR. The effect of the inhibitor on cell proliferation was determined by a cell growth assay. The inhibitor was also administered into bleomycin-treated mice. Histopathological assessment and determination of total collagen levels were carried out to evaluate the severity of lung fibrosis in these mice. Our results demonstrated that treatment with SB 431542 inhibits TGF-β‑induced α-SMA expression in lung fibroblasts, at both the protein and the mRNA levels (P<0.05). However, the inhibitor did not significantly reduce lung fibroblast proliferation. In the bleomycin-induced pulmonary fibrosis mouse model, bleomycin treatment caused important morphological changes, accompanied by an increase in the collagen level of the lungs. Early treatment with SB 431542 prevented the manifestation of histopathological alterations, whereas delayed treatment significantly decreased the collagen level (P<0.05). These results suggest that inhibition of TGF-β signaling, via inhibition of the activin receptor-like kinase-5 (ALK-5) by SB 431542, may attenuate pulmonary fibrosis.

Suppression of autophagy by extracellular vesicles promotes myofibroblast differentiation in COPD pathogenesis

Extracellular vesicles (EVs), such as exosomes and microvesicles, encapsulate proteins and microRNAs (miRNAs) as new modulators of both intercellular crosstalk and disease pathogenesis. The composition of EVs is modified by various triggers to maintain physiological homeostasis. In response to cigarette smoke exposure, the lungs develop emphysema, myofibroblast accumulation and airway remodelling, which contribute to chronic obstructive pulmonary disease (COPD). However, the lung disease pathogenesis through modified EVs in stress physiology is not understood. Here, we investigated an EV-mediated intercellular communication mechanism between primary human bronchial epithelial cells (HBECs) and lung fibroblasts (LFs) and discovered that cigarette smoke extract (CSE)-induced HBEC-derived EVs promote myofibroblast differentiation in LFs. Thorough evaluations of the modified EVs and COPD lung samples showed that cigarette smoke induced relative upregulation of cellular and EV miR-210 expression of bronchial epithelial cells. Using co-culture assays, we showed that HBEC-derived EV miR-210 promotes myofibroblast differentiation in LFs. Surprisingly, we found that miR-210 directly regulates autophagy processes via targeting ATG7, and expression levels of miR-210 are inversely correlated with ATG7 expression in LFs. Importantly, autophagy induction was significantly decreased in LFs from COPD patients, and silencing ATG7 in LFs led to myofibroblast differentiation. These findings demonstrate that CSE triggers the modification of EV components and identify bronchial epithelial cell-derived miR-210 as a paracrine autophagy mediator of myofibroblast differentiation that has potential as a therapeutic target for COPD. Our findings show that stressor exposure changes EV compositions as emerging factors, potentially controlling pathological disorders such as airway remodelling in COPD.

Increased expression of protease nexin-1 in fibroblasts during idiopathic pulmonary fibrosis regulates thrombin activity and fibronectin expression

Idiopathic pulmonary fibrosis (IPF) is a chronic diffuse lung disease characterized by an accumulation of excess fibrous material in the lung. Protease nexin-1 (PN-1) is a tissue serpin produced by many cell types, including lung fibroblasts. PN-1 is capable of regulating proteases of both coagulation and fibrinolysis systems, by inhibiting, respectively, thrombin and plasminergic enzymes. PN-1 is thus a good candidate for regulating tissue remodeling occurring during IPF. We demonstrated a significant increase of PN-1 expression in lung tissue extracts, lung fibroblasts and bronchoalveolar lavage fluids of patients with IPF. The increase of PN-1 expression was reproduced after stimulation of control lung fibroblasts by transforming growth factor-β, a major pro-fibrotic cytokine involved in IPF. Another serpin, plasminogen activator inhibitor-1 (PAI-1) is also overexpressed in fibrotic fibroblasts. Unlike PAI-1, cell-bound PN-1 as well as secreted PN-1 from IPF and stimulated fibroblasts were shown to inhibit efficiently thrombin activity, indicating that both serpins should exhibit complementary roles in IPF pathogenesis, via their different preferential antiprotease activities. Moreover, we observed that overexpression of PN-1 induced by transfection of control fibroblasts led to increased fibronectin expression, whereas PN-1 silencing induced in fibrotic fibroblasts led to decreased fibronectin expression. Overexpression of PN-1 lacking either its antiprotease activity or its binding capacity to glycosaminoglycans had no effect on fibronectin expression. These novel findings suggest that modulation of PN-1 expression in lung fibroblasts may also have a role in the development of IPF by directly influencing the expression of extracellular matrix proteins. Our data provide new insights into the role of PN-1 in the poorly understood pathological processes involved in IPF and could therefore give rise to new therapeutic approaches.

TGFβ1 mediates alcohol-induced Nrf2 suppression in lung fibroblasts.

BackgroundChronic alcohol ingestion induces the expression of transforming growth factor beta-1(TGFβ1), inhibits nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-mediated activation of the antioxidant response element (ARE), depletes alveolar glutathione pools, and potentiates acute lung injury. In this study, we examined the mechanistic relationship between TGFβ1 and Nrf2-ARE signaling in the experimental alcoholic lung.MethodsWild-type mice were treated ± alcohol in drinking water for 8 weeks and their lungs were assessed for Nrf2 expression. In parallel, mouse lung fibroblasts were cultured ± alcohol and treated ± sulforaphane (SFP; an activator of Nrf2), ±TGFβ1, ±TGFβ1 neutralizing antibody, and/or ±activin receptor-like kinase 5 inhibitors (to block TGβ1 receptor signaling) and then analyzed for the expression of Nrf2, Kelch-like ECH-associated protein 1 (Keap1) and TGFβ1, Nrf2-ARE activity, and the expression of the Nrf2-ARE-dependent antioxidants glutathione s-transferase theta 2 (GSTT2) and glutamate-cysteine ligase catalytic subunit (GCLC). Finally, silencing RNA (siRNA) of Nrf2 was then performed prior to alcohol exposure and subsequent analysis of TGFβ1 expression.ResultsAlcohol treatment in vivo or in vitro decreased Nrf2 expression in murine whole lung and lung fibroblasts, respectively. In parallel, alcohol exposure in vitro decreased Keap1 gene and protein expression in lung fibroblasts. Furthermore, alcohol exposure increased TGFβ1 expression but decreased Nrf2-ARE activity and expression of the ARE-dependent genes for GSTT2 and GCLC. These effects of alcohol were prevented by treatment with SFP; in contrast, Nrf2 SiRNA expression exacerbated alcohol-induced TGFβ1 expression. Finally, TGFβ1 treatment directly suppressed Nrf2-ARE activity whereas blocking TGFβ1 signaling attenuated alcohol-induced suppression of Nrf2-ARE activity.ConclusionsAlcohol-induced oxidative stress is mediated by TGFβ1, which suppresses Nrf2-ARE-dependent expression of antioxidant defenses and creates a vicious cycle that feeds back to further increase TGFβ1 expression. These effects of alcohol can be mitigated by activation of Nrf2, suggesting a potential therapy in individuals at risk for lung injury due to alcohol abuse.

Abstract 535: Expression of miR-21 in cancer associated fibroblasts in lung adenocarcinoma

Abstract Lung cancer is the leading cause of cancer mortality worldwide, and novel therapeutic targets and molecular diagnostic markers are highly warranted. Micro RNAs (miRNAs) are a class of small non-coding RNAs associated with a variety of cancer. miRNA-21 is an “oncomiR” and upregulated in many cancers, including lung cancer, and high miR-21 levels have been associated with poor prognosis. To determine the localization of miR-21 and to compare its expression levels, we performed in situ hybridization of miR-21 on 89 mixed subtypes of lung adenocarcinoma. miR-21 was predominantly expressed in cancer associated fibroblasts (CAF)s expressing α-SMA, with no difference in expression between non-invasive and invasive component. In addition, stromal miR-21 expression was significantly associated with decreased overall survival (p=0.0021). In functional analyses of miR-21 in vitro, miR-21 was overexpressed in lung fibroblasts to assess migration and invasion. miR-21 expression in lung fibroblasts promoted migration and invasion. Moreover, the proliferation of lung cancer cells was accelerated by conditioned medium of miR-21-expressing lung fibroblasts. These results suggest that miR-21 overexpression in CAFs of lung adenocarcinoma might serve as a prognostic biomarker and be involved in cancer cell proliferation through interactions with cancer cells. Citation Format: Akiko Kunita, Shigeki Morita, Masashi Fukayama. Expression of miR-21 in cancer associated fibroblasts in lung adenocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 535. doi:10.1158/1538-7445.AM2014-535