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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. Abbreviations Unlabelled Table SAHA suberanilohydroxamic acid TGF-β1 transforming growth factor-β1 COX-2 cyclooxygenase-2 TIA-1 T-cell intracellular antigen-1 PGE2 prostaglandin E2 IPF idiopathic pulmonary fibrosis DAC Decitabine HMT histone methyltransferase EZH2 enhancer of zeste homolog 2 DZNep 3-deazaneplanocin A 3′-UTR 3′-untranslated region α-SMA α-smooth muscle actin ECM extracellular matrix COL1 collagen 1 DNMT DNA methyltransferase HAT histone acetyltransferase HDAC histone deacetylase H3K9me3 histone H3 lysine 9 trimethylation ARE AUUUA-rich element HuR human antigen R ELAV1 ELAV-like RNA binding protein 1 TTP Tristetraprolin CUGBP2 CUG triplet repeat, RNA binding protein 2 F-NL fibroblast from non-fibrotic lung FCS fetal calf serum, Highlights • The HDAC inhibitor SAHA upregulates the expression of the antifibrotic gene COX-2 post-transcriptionally. • The mechanism relies on the downregulation of TIA-1, a negative regulator of COX-2 translation. • SAHA has a therapeutic potential by preventing COX-2 repression induced by TGF-β1 in human lung fibroblasts.

Selective repression of the antifibrotic gene CXCL10 contributes to tissue remodeling in idiopathic pulmonary fibrosis (IPF). We have previously reported that histone deacetylation and histone H3 lysine 9 (H3K9) methylation are involved in CXCL10 repression. In this study, we explored the role of H3K27 methylation and the interplay between the two histone lysine methyltransferases enhancer of zest homolog 2 (EZH2) and G9a in CXCL10 repression in IPF. By applying chromatin immunoprecipitation, Re-ChIP, and proximity ligation assays, we demonstrated that, like G9a-mediated H3K9 methylation, EZH2-mediated histone H3 lysine 27 trimethylation (H3K27me3) was significantly enriched at the CXCL10 promoter in fibroblasts from IPF lungs (F-IPF) compared with fibroblasts from nonfibrotic lungs, and we also found that EZH2 and G9a physically interacted with each other. EZH2 knockdown reduced not only EZH2 and H3K27me3 but also G9a and H3K9me3, and G9a knockdown reduced not only G9 and H3K9me3 but also EZH2 and H3K27me3. Depletion and inhibition of EZH2 and G9a also reversed histone deacetylation and restored CXCL10 expression in F-IPF. Furthermore, treatment of fibroblasts from nonfibrotic lungs with the profibrotic cytokine transforming growth factor-β1 increased EZH2, G9a, H3K27me3, H3K9me3, and histone deacetylation at the CXCL10 promoter, similar to that observed in F-IPF, which was correlated with CXCL10 repression and was prevented by EZH2 and G9a knockdown. These findings suggest that a novel and functionally interdependent interplay between EZH2 and G9a regulates histone methylation-mediated epigenetic repression of the antifibrotic CXCL10 gene in IPF. This interdependent interplay may prove to be a target for epigenetic intervention to restore the expression of CXCL10 and other antifibrotic genes in IPF.

Background: Airway inflammation is a hallmark of asthma disease, and cytokines play a major role in the inflammation process. Human airway smooth muscle cells (HASMCs) are considered as an important source of inflammatory cytokines and chemokines in Asthma. Inhaled corticosteroids (ICS) are used with asthmatic patients as anti-inflammatory therapy to control the severity of the disease and maintain lung function. Clinical studies suggest asthmatic smokers do not respond to steroid as well as non-smokers, but whether cigarette smoke can reduce steroid sensitive in vitro is unclear. We hypothesise that cigarette smoke extract (CSE) reduces the anti-inflammatory effect of fluticasone in HASMCs. Methods: CSE was prepared from the smoke of two cigarettes bubbled into 20 ml of cell culture medium. Confluent and growth-arrested HASMCs were pre-treated with and without CSE (3.5%) for 24 or 48 hours and then were treated with fluticasone (10-11-10-6 M) for 1 hour prior to incubation with TNFa (1 ng/ml), CSE (3.5%), or TNFa + CSE for 24 hours. Cytokines and chemokines were measured in the collected medium by ELISA. MTT assay was used to assess the cell viability. IC50 of fluticasone was calculated using GraphPad Prism. Results: We found that TNFa induced CXCL8 and IL-6 production, but not IL-5, IL-13 and VEGF production, in HASMCs. CSE also induced CXCL8 and IL-6 production. TNFa- and CSE-induced CXCL8 was similarly inhibited by fluticasone in a concentration-dependent manner (IC50 0.2135nM vs 0.5253nM). Co-treatment of TNFa and CSE reduced the inhibitory effect of fluticasone significantly on CXCL8 production compared with TNFa alone (p

Background: Chronic obstructive pulmonary disease (COPD) is characterised by an inflammatory response in the airways to inhaled substances like cigarette smoke. It is known that CSE stimulates the production of a number of inflammatory cytokines and chemokines in HASMCs. E-cigarettes are used to aid smoking cessation but they may also have pro-inflammatory effects. However, the effect of ECVE on cytokine and chemokine production in HASMCs is unknown. This study aims to compare the effect of CSE and ECVE on the production of inflammatory cytokines and chemokines in HASMCs. Methods: CSE (from 2 cigarettes) and ECVE with and without nicotine (N-/N+) (from 6ml e-liquid, 0.24mg/ml nicotine) were prepared by bubbling cigarette smoke and e-cigarette vapour into 20ml of cell culture medium. Confluent and growth-arrested HASMCs were treated with CSE and ECVE for 24 h. Cytokines and chemokines in the medium were measured by ELISA. Cell viability was analysed by MTT assay. Results: 5% CSE and 10% ECVE (N-/N+) reduced HASMC cell viability. CSE (1, 2 and 3.5%) concentration-dependently induced the production of IL-5, IL-6, IL-8, IL-13, vascular endothelial growth factor (VEGF) and CCL2, but not CXCL-10 and RANTES. ECVE (1, 2.5 and 5%) only significantly induced the production of IL-13, but not the others. Conclusion: Our results suggest that cigarette smoke has a wider pro-inflammatory effect in the airway than E-cigarette vapor. The effect of ECVE (N-/N+) on the cytotoxicity and IL-13 production suggests that E-cigarette vapor, not the nicotine in E-cigarette liquid, is harmful to the airway and can also cause airway inflammation.

Introduction Pulmonary Hypertension (PH) is a common and serious complication of COPD. We previously demonstrated that Cigarette Smoke Extracts (CSE) induced imbalanced vasoactive gene expression in human Pulmonary Artery Smooth Muscle Cells (hPASMCs) and Pulmonary Artery Endothelial Cells (hPAECs). Although increasing evidence shows that electronic cigarette (e-cigarette) containing nicotine is associated with increased risk of cardiovascular disease, the effect of e-cigarette vapour extracts (ECVE) on vasoactive gene expression in hPASMCs and hPAECs is unknown. We hypothesize that ECVE may have similar effects as CSE on the induction of an imbalance between excessive vasoconstrictors and deficient vasodilators, which then contributes to aberrant hPASMC proliferation in COPD-associated PH. Methods ECVE was prepared the same way as CSE from 6 ml e-cigarette liquid with and without nicotine (0.24 mg/ml nicotine, total nicotine the same as two cigarettes used for CSE). Confluent hPASMCs and hPAECs were treated with different concentrations of ECVE. Western blotting and real-time RT-PCR were used to assess protein and mRNA expression of Prostacyclin Synthase (PGIS), and Cyclooxygenase-2 (COX-2), Endothelial Nitric Oxide Synthase (eNOS), Thromboxane A Synthase (TXAS), Endothelin 1(ET-1), respectively. Results PGIS protein was markedly reduced by ECVE with and without nicotine in a concentration-dependent manner in hPASMCs. However, ECVE had no effect on the protein of PGIS in hPAECs and mRNA expression of PGIS in both hPASMCs and hPAECs. ECVE with and without nicotine had no effect on the protein and mRNA expression of eNOS, COX-2, and TXAS in both hPASMCs and hPAECs. Although ECVE had no effect on ET-1 mRNA in hPAECs, the mRNA expression of ET-1 was significantly downregulated by ECVE with nicotine. Conclusion Although ECVE had no similar effects on imbalanced vasoactive gene expression as CSE, ECVE reduced the PGIS protein and ET-1 mRNA in hPASMCs, which then could contribute to aberrant hPASMC proliferation in COPD-associated PH. Further studies will be conducted to measure the mediator releases of NO, PGI2, TXA2, PGE2, and ET-1 to confirm the effects of ECVE.

Introduction: Pulmonary Hypertension (PH) is a common and serious complication of COPD. We previously demonstrated that Cigarette Smoke Extracts (CSE) induced imbalanced vasoactive gene expression in human Pulmonary Artery Smooth Muscle Cells (hPASMCs) and Pulmonary artery endothelial cells (hPAECs). Although increasing evidence shows that electronic cigarette (e-cigarette) containing nicotine is associated with increased risk of cardiovascular disease, the effect of e-cigarette vapour extraction (ECVE) on vasoactive gene expression in hPASMCs and hPAECs is unknown. We hypothesize that ECVE may have similar effects as CSE on the induction of an imbalance between excessive vasoconstrictors and deficient vasodilators, which then contributes to aberrant hPASMC proliferation in COPD-associated PH and can be a target for therapeutic intervention. Methods: ECVE was prepared the same way as CSE from 6 ml e-cigarette liquid (0.24 mg/ml nicotine, total nicotine the same as two cigarettes used for CSE). Confluent hPASMCs and hPAECs were treated with different concentrations of ECVE. Western blotting and real-time RT-PCR were used to assess protein and mRNA expression of Prostacyclin Synthase (PGIS), and Cyclooxygenase-2 (COX-2), Endothelial Nitric Oxide Synthase (eNOS), Thromboxane A Synthase (TXAS), Endothelin 1(ET-1), respectively. Results: ECVE reduced the protein and mRNA expression of eNOS and PGIS and upregulated COX-2 and TXAS level in a concentration-dependent manner. Interestingly, ECVE did not have any effect on ET-1. Conclusion: Our findings suggest that ECVE may have similar effects on imbalanced vasoactive gene expression as CSE.

COPD is a common chronic inflammatory lung disease with cigarette smoke (CS) as the major cause. The receptor for advanced glycation end products (RAGE) and its ligands are critically involved in inflammation. Inhaled corticosteroids are poorly effective in improving the symptoms of COPD. This underlines the need for new anti-inflammatory therapies. Cordycepin is a purine nucleoside antimetabolite. Simvastatin is a lipid-lowering agent. But the effect of Cordycepin and Simvastatin on CSE-induced inflammatory gene expression in human bronchial epithelial cells has not been explored. Human BEAS-2B bronchial epithelial cells were treated with Cordycepin + CSE (2.5%) and Simvastatin + CSE (2.5%). IL-8 andIL-6 production was measured by ELISA. RAGE and RAGE ligand HMBG-1 expression was analysed by Western blot. We found that CSE-induced IL-8 production was inhibited by Cordycepin and Simvastatin but IL-6 production was enhanced by Cordycepin and not affected by Simvastatin. CSE and both drugs had no effect on basal RAGE and HMGB-1 expression. Since IL-6 contribute to the pathogenesis of COPD by modulating pulmonary function but not necessarily inflammation and neutrophil-driven airway inflammation (by IL-8 and other chemokines) is critically involved in the pathogenesis of COPD, our findings suggest cigarette smoke, by inducing inflammatory responses from human bronchial epithelial cells, may play an important role in airway inflammation in COPD and that Cordycepin and Simvastatin, by reducing IL-8 production (unlikely via RAGE inhibition), may have therapeutic potential in COPD.

Background Chronic obstructive pulmonary disease (COPD) is an inflammatory disorder of the respiratory tract characterised by airflow obstruction. It is increasingly recognised that the innate immune pattern-recognition receptors may contribute to airway inflammation in COPD in response to environmental factors such as cigarette smoke (CS). One pattern-recognition receptor that has recently come to attention in chronic airway disease is the cell surface receptor for advanced glycation end products (RAGE). RAGE also exists as a soluble form (sRAGE) that primarily functions as receptor decoy and an endogenous inhibitor of RAGE signalling. Clinical studies show that smokers with or without COPD have significantly greater levels of RAGE expression in airway epithelial cells compared with never smokers. However, the role of RAGE in mediating CS-induced inflammatory gene expression has not been understood. We hypothesise that CS can induce RAGE expression, sRAGE reduction, and inflammatory gene expression in human bronchial epithelial cells (BEAS-2B). Method Confluent BEAS-2B cells were treated with different concentrations of Cigarette Smoke Extract (CSE) (1%, 2.5%, and 5%) for 24 hours. Western blotting was used to assess protein expression of RAGE in cell lysate. ELISA was used to measure interleukin 6 (IL-6), CXCL1 (GRO-α), CXCL5 (ENA-78), CXCL8 (IL-8), CXCL10 (IP-10), CCL11 (eotaxin), and sRAGE in culture medium. Result We found that IL-6 and CXCL8 releases were markedly increased by CSE in a concentration-dependant manner, but CXCL-1, CXCL5, CXCL10 and CCL11 could not be detected in both untreated and CSE-treated cells. Interestingly, RAGE was highly expressed in untreated cells and CSE treatment did not further increase its expression. Furthermore, sRAGE was also undetectable in both untreated and CSE-treated cells. Conclusion These findings suggest that CSE can induce inflammatory gene expression in BEAS-2B cells. Further experiments are being conducted to explore the effect of CSE on other inflammatory gene expression and to investigate the role of RAGE in mediating CSE-mediated inflammatory response in BEAS-2B cells.

Background Pulmonary Hypertension (PH) is a common and serious complication of Chronic Obstructive Pulmonary Disease (COPD) associated with increased mortality and morbidity and characterised by Pulmonary Artery Smooth Muscle Cell (PASMC) hyperproliferation and vascular remodelling. Studies suggest that chronic hypoxia and Cigarette Smoke (CS) can cause aberrant PASMC proliferation and vascular remodelling, however, how cigarette smoke and hypoxia contribute to pulmonary artery wall thickening and PH in COPD is not fully understood. We hypothesise that hypoxia and CS can induce an imbalance between excessive vasoconstrictors and deficient vasodilators, which then contribute to aberrant PASMC proliferation in COPD-associated PH and can be a target for therapeutic intervention. Method To prove the hypothesis, confluent Human Pulmonary Artery Smooth Muscle Cells (hPASMCs) and Human Pulmonary Artery Endothelial Cells (hPAECs) were treated with different concentrations of Cigarette Smoke Extract (CSE) (1%, 2.5%, and 5%) under normoxic (21% O 2 ) or hypoxic (1% O 2 ) condition for 72 hour. The protein and mRNA expression of Prostacyclin Synthase (PGIS), Cyclooxygenase-2 (COX-2), Endothelial Nitric Oxide Synthase (eNOS), Thromboxane A Synthase (TXAS), and Endothelin 1(ET-1) was analysed by Western blotting and real-time RT-PCR, respectively. Results The expression of vasodilator genes eNOS and PGIS was noticeably downregulated in both hPASMCs and hPAECs, whereas TXAS and COX-2 expression was markedly induced by CSE and hypoxia, either individually or in combination in both hPASMCs and hPAECs. ET-1 expression was increased by CSE and hypoxia in hPAECs. Interestingly, ET-1 was upregulated by hypoxia, but reduced by CSE, with a net increase when both were combined in hPASMCs. Conclusion These findings support our hypothesis that CS and hypoxia can cause an imbalance between excessive vasoconstrictors and deficient vasodilators in hPASMC and hPAECs. This imbalance may eventually lead to aberrant PASMC proliferation and vascular remodelling in COPD-associated PH. Further experiments are being conducted to confirm this by analysing vasoactive gene expression and mediator release in both hPASMCs and hPAECs. Our findings also pave the way for further studies on cellular functions and intervention drug effects.

Chemokine (CC motif) ligand-2 (CCL2) is a member of C-C chemokine superfamily that contributes to inflammatory and fibrotic process. Studies in patients and experimental animals provide compelling evidence that increased CCL2 expression plays an important role in the development of fibroproliferative lung disease. The up-regulated CCL2 expression in pulmonary fibrosis is also involved in the potent profibrotic effects that thrombin exerts during lung injury. Here, we investigated the transcriptional mechanism involved in CCL2 production by thrombin in human primary lung fibroblasts and explored the transcriptional mechanism of increased CCL2 expression in pulmonary fibrosis. Thrombin increased CCL2 mRNA levels but not mRNA stability, suggesting it was acting transcriptionally. The increased binding of transcription factors to nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) elements in the CCL2 promoter contributed to active transcription following thrombin stimulation. Primary human lung fibroblasts isolated from patients with idiopathic pulmonary fibrosis (IPF) produced significantly higher levels of CCL2 than nonfibrotic lung fibroblasts. Furthermore, chromatin immunoprecipitation assays detected increased binding of NF-κB p65 and AP-1 subunit c-Jun to the CCL2 promoter of IPF cells both in the presence and absence of thrombin stimulation. The significantly increased binding of p65 and c-Jun to the CCL2 promoter was also observed in the lung tissue of bleomycin-induced pulmonary fibrosis murine model. Collectively, these findings strongly suggest that the increased binding of transcription factors to NF-κB and AP-1 elements in the CCL2 promoter is responsible for the active transcription expression of CCL2 in pulmonary fibrosis.

Introduction and objectives: Idiopathic Pulmonary Fibrosis (IFP) is a fatal interstitial lung disease with unknown aetiology. Lung myofibroblasts (activated fibrobalsts) are the major effector cells in the pathogenesis of IPF. Transforming growth factor-β (TGF-β1) is a potent activator of fibroblasts. Lack of effective treatment options necessitates novel therapeutic approaches. Epigenetic drugs, by inhibiting chromatin modifying enzymes involved in gene expression control, represent promising agents capable of modulating the cellular phenotype. We previously demonstrated that the cyclooxygenase-2 (COX-2) gene is epigenetically silenced in lung fibroblasts from IPF patients (F-IPF)1 and epigenetic inhibitors and restore COX-2 expression. However, whether epigenetic inhibitors can alter fibroblast phenotype remains unknown. This study aimed to investigate the effect of four different epigenetic enzyme inhibitors on fibroblast phenotype change in IPF. Methods: F-IPF and fibroblasts from non-fibrotic lung (F-NL) treated with TGF-β1 were cultured to test the effects of the epigenetic inhibitors BIX01294 (BIX, G9a histone methyltransferase inhibitor), 3-deazaneplanocin A (DZNep, EZH2 histone methyltransferase inhibitor), SAHA (histone deacetylases inhibitor) and Decitabine (DAC, DNA demethylating agent), in comparison with the COX-2 products prostaglandin E2 (PGE2). The expression of COX-2 and myofibroblast markers collagen 1 (COL1) and α-smooth muscle actin (α-SMA) was assessed. The COX-2 DNA promoter methylation level was analysed by bisulfite sequencing. Results: TGF-β1 induced a myofibroblast phenotype in F-NL characterised by COL1 and α-SMA upregulation and COX-2 downregulation, similar to F-IPF. PGE2 and SAHA were able to maintain/restore COX-2 expression in TGF-β1-induced myofibroblasts and F-IPF. DAC demonstrated similar effect in TGF-β1 treated F-NL only. SAHA also reduced COL1 and α-SMA expression. But DZNep and BIX showed no effect. No differences in the COX-2 promoter methylation was detected between F-NL and F-IPF. Conclusions: Among the epigenetic inhibitors tested, SAHA shows a promising antifibrotic effect by inhibiting fibroblast activation and the underlying molecular mechanisms are currently under investigation.

Severe asthma is associated with airway remodeling, characterized by structural changes including increased smooth muscle mass and matrix deposition in the airway, leading to deteriorating lung function. TGF-β is a pleiotropic cytokine leading to increased synthesis of matrix molecules by human airway smooth muscle (HASM) cells and is implicated in asthmatic airway remodeling. TGF-β is synthesized as a latent complex, sequestered in the extracellular matrix, and requires activation for functionality. Activation of latent TGF-β is the rate-limiting step in its bioavailability. This study investigated the effect of the contraction agonists LPA and methacholine on TGF-β activation by HASM cells and its role in the development of asthmatic airway remodeling. The data presented show that LPA and methacholine induced TGF-β activation by HASM cells via the integrin αvβ5. Our findings highlight the importance of the β5 cytoplasmic domain because a polymorphism in the β5 subunit rendered the integrin unable to activate TGF-β. To our knowledge, this is the first description of a biologically relevant integrin that is unable to activate TGF-β. These data demonstrate that murine airway smooth muscle cells express αvβ5 integrins and activate TGF-β. Finally, these data show that inhibition, or genetic loss, of αvβ5 reduces allergen-induced increases in airway smooth muscle thickness in two models of asthma. These data highlight a mechanism of TGF-β activation in asthma and support the hypothesis that bronchoconstriction promotes airway remodeling via integrin mediated TGF-β activation.

CXCL8 is a neutrophil and mast cell chemoattractant that is involved in regulating inflammatory cell influx in asthma. Here, we investigated the transcriptional mechanism involved in CXCL8 induction by TNF-α in cultured human airway smooth muscle (HASM) cells and compared these in cells from nonasthmatic and asthmatic individuals. Transfection studies with mutated CXCL8 promoter constructs identified NF-κB, activating protein-1, and CAAT/enhancer binding protein (C/EBP)β as key transcription factors, and binding of these three transcription factors to the CXCL8 promoter after TNF-α stimulation was confirmed by chromatin immunoprecipitation analysis. Cells derived from asthmatic individuals produced significantly higher levels of CXCL8 than nonasthmatic cells both basally and following 24 h of stimulation with TNF-α (p < 0.001). Furthermore, chromatin immunoprecipitation studies detected increased binding of NF-κB p65 and RNA polymerase II to the CXCL8 promoter of asthmatic HASM cells both in the presence and absence of TNF-α stimulation. This was not due to either an increased activation or phosphorylation of NF-κB per se or to an increase in its translocation to the nucleus. Increased binding of C/EBPβ to the CXCL8 promoter of unstimulated cells was also detected in the asthmatic HASM cells. Collectively these studies show that HASM cells from asthmatic individuals have increased CXCL8 production due to the presence of a transcription complex on the CXCL8 promoter, which contains NF-κB, C/EBPβ, and RNA polymerase II. This is the first description of an abnormality in transcription factor binding altering chemokine expression in airway structural cells in asthma.

Transforming growth factor beta (TGFbeta) is a key remodelling factor in asthma. It is produced as a latent complex and the main limiting step in TGFbeta bioavailability is its activation. Mast cell tryptase has been shown to stimulate the release of functionally active TGFbeta from human airway smooth muscle (ASM) cells [P. Berger, P.O. Girodet, H. Begueret, O. Ousova, D.W. Perng, R. Marthan, A.F. Walls, J.M. Tunon de Lara, Tryptase-stimulated human airway smooth muscle cells induce cytokine synthesis and mast cell chemotaxis, FASEB J. 17 (2003) 2139-2141]. The aim of this study was to determine if tryptase could cause TGFbeta activation as well as expression in ASM cells via its receptor, proteinase-activated receptor 2 (PAR2). Tryptase caused TGFbeta activation without affecting levels of total TGFbeta. This effect was inhibited by the selective tryptase inhibitor FUT175 and leupeptin but not mimicked by the PAR2 activating peptide SLIGKV-NH(2). Furthermore, the ASM cells used in the study did not express PAR2. The results indicate that tryptase activates TGFbeta via a PAR2-independent proteolytic mechanism in human ASM cells and may help understanding the role of tryptase in asthma.

Here we tested the effect of interleukin-1β, a pro-inflammatory cytokine, on cAMP accumulation and chloride efflux in Calu-3 airway epithelial cells in response to ligands binding to adenylyl cyclase-coupled receptors such as the β2 adrenoreceptor and EP prostanoid receptors. Interleukin-1β significantly increased isoprenaline-induced cAMP accumulation by increasing β2 adrenoreceptor numbers via a protein kinase A-dependent mechanism. In contrast, interleukin-1β significantly impaired prostaglandin E2-induced cAMP accumulation by induction of cyclo-oxygenase-2, prostaglandin E2 production, and a resulting down-regulation of adenylyl cyclase. The cAMP changes were all mirrored by alterations in chloride efflux assessed using the fluorescent chloride probe N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide with interleukin-1β increasing chloride efflux in response to isoprenaline and reducing the response to prostaglandin E2. Studies with glibenclamide confirmed that chloride efflux was via the cystic fibrosis transmembrane conductance regulator. Calu-3 expresses EP4 receptors, but not EP2, and receptor expression is reduced by interleukin-1β. Collectively, these results provide mechanistic insight into how interleukin-1β can differentially regulate cAMP generation and chloride efflux in response to different adenylyl cyclase-coupled ligands in the same cell. These findings have important implications for diseases involving inflammation and abnormal ion flux such as cystic fibrosis.

Chemokine-mediated inflammatory cell infiltration is a hallmark of asthma. We recently demonstrated that glucocorticoids and β2-agonists additively or synergistically suppress tumor necrosis factor-α (TNFα)-induced production of chemokines eotaxin and interleukin-8 (IL-8), respectively, in human airway smooth muscle (HASM) cells, which may partly explain their combined benefits in asthma. Peroxisome proliferator-activated receptors (PPARs) also modulate inflammatory gene expression. We reported here that the PPARγ agonists 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2) and troglitazone, but not PPARα agonist WY-14643, inhibited TNFα-induced production of eotaxin and monocyte chemotactic protein-1 (MCP-1) but not IL-8. Eotaxin inhibition was transcriptional and additively enhanced by the glucocorticoid fluticasone and the β2-agonist salmeterol, whereas MCP-1 inhibition was post-transcriptional and additively and synergistically enhanced by fluticasone and salmeterol, respectively. Coimmunoprecipitation revealed that 15d-PGJ2 induced a protein-protein interaction between PPARγ and the glucocorticoid receptor (GR) in TNFα-treated HASM cells, which was enhanced by fluticasone and salmeterol. 15d-PGJ2, fluticasone, and salmeterol all inhibited TNFα-induced histone H4 acetylation at the eotaxin promoter and NF-κB p65 binding to the eotaxin promoter and induced PPARγ and GR association with the eotaxin promoter, as analyzed by chromatin immunoprecipitation assay. Our data suggest that chemokine expression in HASM cells is differentially regulated by PPARγ agonists and that the interaction between PPARγ and GR may be responsible for the additive and synergistic inhibition of chemokine expression by PPARγ agonists, glucocorticoids, and β2-agonists, particularly the chromatin-dependent suppression of eotaxin gene transcription. The interaction may have wide applications and may provide a potential target for pharmacological and molecular intervention.

Evidence is accumulating to suggest that the inducible isoenzyme of cyclooxygenase (COX)-2 is up-regulated in human cancers and epidemiological studies indicate that COX inhibitors may have a protective effect on the development of lung cancer. We used immunohistochemistry and Western blotting to investigate COX expression in lung tumour specimens and three lung cancer cell lines. Sixty-five archival lung tissue samples, including 46 squamous cell and 6 adenocarcinoma lung resections, and 13 small cell lung cancer (SCLC) biopsies were studied. Dense and intense cytoplasmic COX-2 staining was found in all 52 resections from non-small cell lung cancer (NSCLC). The staining was diffuse and much stronger than adjacent respiratory epithelium. COX-2 staining was relatively weak in the majority of the SCLC samples. The bronchial and bronchiolar epithelium in the surrounding normal lung structures showed uniform COX immunoreactivity with apical concentration of the stain. There was no increase in COX-1 staining in any tumour type. Western blot analysis of the cancer lines revealed significantly higher expression of COX-1 in CORL23 line and COX-2 in two NSCLC cell lines (MOR/P; A549) compared with the expression of COX-1 and COX-2 in cultured normal bronchial epithelial cells. Our findings demonstrated COX-2 overexpression in NSCLC.

Bradykinin and interleukin-1beta (IL-1beta) induce cyclooxygenase 2 (COX-2) in human airway smooth muscle cells. Here we extended our study to explore the gene transcriptional regulation. By transfection with various COX-2 promoter reporter constructs, we found that the bp -327-to-+59 promoter region was essential for COX-2 gene transcription by bradykinin and IL-1beta and that the cyclic AMP response element (CRE) was critical in bradykinin-induced transcription, whereas nuclear factor IL-6 and CRE and, to a lesser extent, nuclear factor-kappaB (NF-kappaB) were involved in IL-1beta-induced transcription. An electrophoretic mobility shift assay revealed that both bradykinin and IL-1beta elicited CRE-binding protein-1 (CREB-1) binding, and IL-1beta also elicited CCAAT/enhancer-binding protein beta and NF-kappaB binding to their respective elements in the COX-2 promoter. These transcription factors were associated with the COX-2 promoter, which was dynamically linked to different patterns of histone H4 acetylation by bradykinin and IL-1beta, as demonstrated by chromatin immunoprecipitation. We also revealed that endogenous prostaglandin E(2) was critical in bradykinin-induced COX-2 transcription initiation and involved in IL-1beta-induced COX-2 transcription at a latter stage. Our result provide the first evidence that COX-2 transcriptional regulation by different stimuli involves different promoter elements and transcription factors and is associated with chromatin remodeling after selective histone H4 acetylation in a stimulus-specific manner.

Bradykinin (BK) is a potent neutrophil chemotractant, proinflammatory mediator, and angiogenic factor, which acts through G protein-coupled receptors (GPCRs). Here we studied the mechanisms involved in IL-8 generation by BK in human airway smooth muscle cells focusing on the transcription factors involved and role of endogenous prostanoids in transcription factor activation. Transfection experiments with wild-type IL-8 promoter constructs or constructs with NF-kappaB, AP-1, and NF-IL-6 binding site mutations suggested that all three transcription factors were necessary for optimal IL-8 expression. BK increased NF-kappaB, AP-1, and NF-IL-6 binding to the IL-8 promoter by electrophoretic mobility shift assay. NF-kappaB, the most important transcription factor in the current study, was translocated to the nucleus after BK stimulation. Indomethacin, a cyclooxygenase inhibitor, partially inhibited IL-8 release and the promoter binding of AP-1 and NF-IL-6, but not NF-kappaB. Furthermore, exogenous prostaglandin E2 stimulated AP-1 and NF-IL-6 binding to the IL-8 promoter. The anti-inflammatory glucocorticoid dexamethasone inhibited NF-kappaB translocation and the promoter binding of NF-kappaB, AP-1, and NF-IL-6. These results are the first to delineate the transcription factors involved in BK induced IL-8 release. Transcriptional activation of the IL-8 promoter by BK involves the prostanoid-independent activation of NF-kappaB, and prostanoid-dependent activation of AP-1 and NF-IL-6 plays a key role in augmenting the response. Endogenous prostanoid generation in response to GPCR ligands such as BK may be an important mechanism whereby GPCRs signal to the nucleus to maximize the transcription of inflammatory response genes.

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been shown to modulate cyclooxygenase (COX)-2 expression, but the mechanisms involved are controversial and may be cell specific. We show in this study that indomethacin (Indo), flurbiprofen (Flur), and the selective COX-2 inhibitor NS-398 induced COX-2 expression and markedly enhanced IL-1beta-induced COX-2 expression in human airway smooth muscle (HASM) cells. These effects were not reversed by exogenous PGE(2), suggesting that they are prostanoid-independent. Indeed, PGE(2) also induced and enhanced IL-1beta-induced COX-2 expression. Peroxisome proliferator-activated receptor (PPAR) alpha and PPARgamma (not PPARbeta) were expressed in HASM cells. PPARgamma activators ciglitizone (Cig) and 15-Deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)), but not the PPARalpha activator WY-14643, mimicked the effect of NSAIDs on COX-2 expression. Treatment with Flur, NS-398, Cig, and 15d-PGJ(2) alone, but not Indo and WY-14643, elevated COX activity; however, neither enhanced IL-1beta-induced COX activity. Pretreatment with dexamethasone suppressed COX-2 expression, PGE(2) release, and COX activity induced by NS-398, Cig, IL-1beta, alone or in combination. Unlike IL-1beta, NS-398 and Cig did not cause NF-kappaB (p65) nuclear translocation, nor did they further enhance IL-1beta-induced NF-kappaB translocation, but they stimulated PPARgamma translocation. Indo, NS-398, Flur, and 15d-PGJ(2), but not WY-14643, induced transcriptional activity of a COX-2 reporter construct containing the peroxisome proliferator response element (PPRE) on their own and enhanced the effect of IL-1beta, but had no effect on a COX-2 reporter construct lacking the PPRE. The results suggest that COX-2 expression by NSAIDs is biologically functional, prostanoid-independent, and involves PPARgamma activation, and provide the first direct evidence that the PPRE in the promoter is required for NSAID-induced COX-2 expression.

Prostanoids are major regulators of smooth muscle function that are generated by cyclooxygenase (COX). Here we hypothesized that cytokines and mediators that regulate the pulmonary circulation would alter COX expression and prostanoid generation in pulmonary artery smooth muscle cells. Bradykinin, transforming growth factor-β1 (TGF-β1), and interleukin-1β (IL-1β) increased inducible COX-2 expression and prostaglandin E2 (PGE2) release. Transfection studies using a COX-2 promoter construct demonstrated that all three agents acted transcriptionally. Constitutive COX-1 protein expression was unchanged. The COX inhibitor indomethacin, the COX-2 inhibitor NS-398, the protein synthesis inhibitor cycloheximide, and the glucocorticoid dexamethasone abrogated the increased PGE2levels. Dexamethasone and cycloheximide prevented COX-2 induction. Hypoxia (3% O2-5% CO2-92% N2) for 24 h selectively augmented TGF-β1-stimulated PGE2 production and COX-2 induction but had no effect alone. Prolonged hypoxic culture alone for 48 and 72 h enhanced COX-2 induction and increased PGE2. These studies show that a number of stimuli are capable of inducing COX-2 in pulmonary artery smooth muscle cells. The interaction between hypoxia and TGF-β1 may be particularly relevant to pulmonary hypertension.

Interleukin (IL)-8, the C-X-C chemokine, is a potent neutrophil chemoattractant that has been implicated in a number of inflammatory airway diseases such as cystic fibrosis. Here we tested the hypothesis that bradykinin, an inflammatory mediator and chloride secretagogue, would increase IL-8 generation in airway epithelial cells through autocrine generation of endogenous prostanoids. Bradykinin increased IL-8 generation in both a non-cystic fibrosis (A549) and cystic fibrosis epithelial cell line (CFTE29[Formula: see text]) that was inhibited by the nonselective cyclooxygenase (COX) inhibitor indomethacin and the COX-2 selective inhibitor NS-398. COX-2 was the only isoform of COX expressed in both cell lines. Furthermore, the COX substrate arachidonic acid and exogenous prostaglandin E2 both increased IL-8 release in A549 cells. These results suggest that bradykinin may contribute to neutrophilic inflammation in the airway by generation of IL-8 from airway epithelial cells. The dependence of this response on endogenous production of prostanoids by COX-2 suggests that selective COX-2 inhibitors may have a role in the treatment of airway diseases characterized by neutrophilic inflammation such as cystic fibrosis or chronic obstructive pulmonary disease.

Selective silencing of the cyclooxygenase-2 (COX-2) gene with the loss of the antifibrotic mediator prostaglandin E2 contributes to the fibrotic process in idiopathic pulmonary fibrosis (IPF). This study explored the role of G9a- and enhancer of zeste homolog 2 (EZH2)-mediated methylation of histone H3 lysine 9 (H3K9me3) and histone H3 lysine 27 (H3K27me3) in COX-2 silencing in IPF. Chromatin immunoprecipitation (ChIP) and re-ChIP assays demonstrated marked increases in H3K9me3, H3K27me3, and DNA methylation, together with their respective modifying enzymes G9a, EZH2, and DNA methyltransferases (Dnmts) and respective binding proteins heterochromatin protein 1 (HP1), polycomb protein complex 1 (PRC1) and methyl CpG binding protein 2 (MeCP2), at the COX-2 promoter in lung fibroblasts from patients with IPF (F-IPFs) compared with fibroblasts from nonfibrotic lungs. HP1, EZH2, and MeCP2 in turn were associated with additional repressive chromatin modifiers in F-IPFs. G9a and EZH2 inhibitors and small interfering RNAs and the Dnmt1 inhibitor markedly reduced H3K9me3 (49−79%), H3K27me3 (44−81%), and DNA methylation (61−97%) at the COX-2 promoter. These reductions were correlated with increased histone H3 and H4 acetylation, resulting in COX-2 mRNA and protein reexpression in F-IPFs. Our results support a central role for G9a- and EZH2-mediated histone hypermethylation and a model of bidirectional, mutually reinforcing, and interdependent crosstalk between histone hypermethylation and DNA methylation in COX-2 epigenetic silencing in IPF.—Coward, W. R., Feghali-Bostwick, C. A., Jenkins, G., Knox, A. J., Pang, L. A central role for G9a and EZH2 in the epigenetic silencing of cyclooxygenase-2 in idiopathic pulmonary fibrosis.

Bronchial vascular remodeling is an important feature of the pathology of chronic asthma, but the responsible mechanisms and main sources of angiogenic factors are unclear. Here we report that human airway smooth muscle cells express vascular endothelial growth factor (VEGF)121, 165, 189, 206 splice variants and secrete VEGF protein constitutively. VEGF protein secretion was increased by the proinflammatory asthma mediator bradykinin through post-transcriptional mechanisms. Bradykinin-induced VEGF secretion was dependent on the B2 bradykinin receptor, activation of protein kinase C, and generation of endogenous prostanoids. This is the first report that bradykinin can increase VEGF secretion in any biological system and the first to show that airway smooth muscle cells produce VEGF. Our results suggest a novel role for human airway smooth muscle in contributing to bronchial mucosal angiogenesis in chronic asthma by secretion of VEGF and suggest a wider role for mesenchymal cell products in mediating angiogenesis in inflammatory and allergic diseases.

1 Airway smooth muscle (ASM) in human asthma shows reduced relaxation and cyclic AMP generation in response to β-adrenoceptor agonists. IL-β attenuates cyclic AMP generation but the underlying mechanism is unclear. We have reported that IL-1β induces cyclo-oxygenase-2 (COX-2) in human ASM cells and results in a marked increase in prostanoid generation with PGE2 and PGI2 as the major products. 2 We investigated the role of COX-2 induction and prostanoid release (measured as PGE2) in IL-1β induced attenuation of cyclic AMP generation in response to the β-adrenoceptor agonist isoprenaline (ISO). 3 Pre-treatment of human ASM cells with IL-1β significantly attenuated cyclic AMP generation in response to high concentrations of ISO (1.0–10.0 μm) in a time- and concentration-dependent manner. The effect was accompanied by a high concentration of PGE2 release. The non-selective COX inhibitor indomethacin (Ind), the selective COX-2 inhibitor NS-398, the protein synthesis inhibitors cycloheximide (CHX) and actinomycin D and the steroid dexamethasone (Dex) all abolished the PGE2 release and prevented the attenuated cyclic AMP generation. 4 COX substrate arachidonic acid time- and concentration-dependently mimicked IL-1β induced attenuation and the effect was prevented by the non-selective COX inhibitors Ind and flurbiprofen, but not by NS-398, CHX and Dex. 5 In contrast to IL-1β, TNFα and IFNγ, which are ineffective in inducing COX-2 and releasing PGE2 from human ASM cells, did not affect the cyclic AMP formation. 6 Our study demonstrates that COX-2 induction and the consequent release of prostanoids plays a crucial role in IL-1β induced attenuation of human ASM cell cyclic AMP response to ISO. British Journal of Pharmacology (1998) 125, 1320–1328; doi:10.1038/sj.bjp.0702193

Prostanoids may be involved in bradykinin (BK)-induced bronchoconstriction in asthma. We investigated whether cyclooxygenase (COX)-2 induction was involved in prostaglandin (PG) E2release by BK in cultured human airway smooth muscle (ASM) cells and analyzed the BK receptor subtypes responsible. BK stimulated PGE2release, COX activity, and COX-2 induction in a concentration- and time-dependent manner. It also time dependently enhanced arachidonic acid release. In short-term (15-min) experiments, BK stimulated PGE2generation but did not increase COX activity or induce COX-2. In long-term (4-h) experiments, BK enhanced PGE2release and COX activity and induced COX-2. The long-term responses were inhibited by the protein synthesis inhibitors cycloheximide and actinomycin D and the steroid dexamethasone. The effects of BK were mimicked by the B2-receptor agonist [Tyr(Me)8]BK, whereas the B1agonist des-Arg9-BK was weakly effective at high concentrations. The B2antagonist HOE-140 potently inhibited all the effects, but the B1antagonist des-Arg9,(Leu8)-BK was inactive. This study is the first to demonstrate that BK can induce COX-2. Conversion of increased arachidonic acid release to PGE2by COX-1 is mainly involved in the short-term effect, whereas B2receptor-related COX-2 induction is important in the long-term PGE2release.

Increased levels of several pro-inflammatory cytokines including interleukin-1β (IL-1β) and tumour necrosis factor-α (TNFα) have been found in bronchoalveolar lavage fluid from symptomatic asthmatic patients. IL-1β, TNFα and interferon-γ (IFNγ) are known to stimulate a number of cells to produce inflammatory mediators such as prostaglandins. Although airway smooth muscle (ASM) is known to be a rich source of prostaglandins, the regulation of cyclo-oxygenase (COX) isoforms and prostanoid production by proinflammatory cytokines have not been studied in human airway smooth muscle. We studied the effects of IL-1β, TNFα and IFNγ on the induction of two isoforms of cyclo-oxygenase and its relation to prostaglandin E2 (PGE2) release and COX activity (reflected by PGE2 synthesis from exogenous arachidonic acid) in human cultured airway smooth muscle cells. IL-1β, but not TNFα or IFNγ, caused a time- and concentration-dependent enhancement in PGE2 and other prostanoid (6-keto-PGF1α, PGF2α, thromboxane B2 (TXB2) and PGD2) production, with PGE2 and 6-keto-PGF1α as the principal products. This stimulation was accompanied by a corresponding increase in COX activity. COX-2 protein measured by Western blot analysis was not detectable in untreated cells, but was increased in a time- and concentration-dependent manner by IL-1β, but not TNFα or IFNγ. In contrast, no variation in the expression of COX-1 protein was observed. Pretreatment with the conventional non-steroidal anti-inflammatory drugs (NSAIDs), indomethacin and ibuprofen, and the selective COX-2 inhibitors, NS-398 and nimesulide, completely blocked IL-1β-induced PGE2 release and COX activity. The glucocorticosteroid dexamethasone and protein synthesis inhibitors, cycloheximide and actinomycin D, not only markedly inhibited IL-1β-stimulated PGE2 release and COX activity but also suppressed IL-1β-induced COX-2 induction. This study demonstrates that human cultured ASM cells release prostanoids in response to IL-1β stimulation and that the response is mostly mediated by the induction of COX-2 rather than COX-1 isoenzyme, implying that airway smooth muscle may be an important source of prostaglandins in human airways and that COX-2 may play an important role in the regulation of the inflammatory process in asthma.

The enhanced nitric oxide (NO) and prostaglandin (PG) generation of activated macrophages is controlled by glucocorticoid-sensitive inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), respectively. Negative feedback regulation of iNOS expression by the products of both pathways has been suggested, but their effects on COX-2 expression have not been examined. We have investigated the effect of E- and I-series prostaglandins that activate adenylate cyclase (AC), forskolin (a direct activator of AC), and other agents that influence the cyclicAMP/cyclicGMP systems on the ability of E. coli endotoxin (lipopolysaccharide, LPS) to induce iNOS and COX-2 in the murine macrophage cell line J774. After a 2-hr pretreatment before adding endotoxin, PGE2, PGI2, forskolin, IBMX (isobutylmethylxanthine, a cyclicAMP/cyclicGMP phosphodiesterase inhibitor), 8-bromo cyclicAMP, and arachidonic acid itself all inhibited the expression of both iNOS and COX-2 (as shown by Western blotting), and reduced NO release and COX activity, whereas PGF2α and 8-bromo cyclic GMP were only weakly effective. The effects of PGE2, PGI2, and forskolin were enhanced by cotreatment with IBMX. The suppression of LPS-induced iNOS induction by PGE2 was functionally significant, in that it protected against the mild cytotoxicity of the NO generated in response to endotoxin. These results provide the first direct evidence for the feedback regulatory suppression of COX-2 induction by a PG-driven cAMP-mediated process, and show that the modulation of iNOS and COX-2 induction shares common features. They also suggest that such modulation is normally held in check by high phosphodiesterase activity within these cells.

Cordycepin (3′ deoxyadenosine) has long been used in the study of in vitro assembled polyadenylation complexes, because it terminates the poly(A) tail and arrests the cleavage complex. It is derived from caterpillar fungi, which are highly prized in Chinese traditional medicine. Here we show that cordycepin specifically inhibits the induction of inflammatory mRNAs by cytokines in human airway smooth muscle cells without affecting the expression of control mRNAs. Cordycepin treatment results in shorter poly(A) tails, and a reduction in the efficiency of mRNA cleavage and transcription termination is observed, indicating that the effects of cordycepin on 3′ processing in cells are similar to those described in in vitro reactions. For the CCL2 and CXCL1 mRNAs, the effects of cordycepin are post-transcriptional, with the mRNA disappearing during or immediately after nuclear export. In contrast, although the recruitment of RNA polymerase II to the IL8 promoter is also unaffected, the levels of nascent transcript are reduced, indicating a defect in transcription elongation. We show that a reporter construct with 3′ sequences from a histone gene is unaffected by cordycepin, while CXCL1 sequences confer cordycepin sensitivity to the reporter, demonstrating that polyadenylation is indeed required for the effect of cordycepin on gene expression. In addition, treatment with another polyadenyation inhibitor and knockdown of poly(A) polymerase α also specifically reduced the induction of inflammatory mRNAs. These data demonstrate that there are differences in the 3′ processing of inflammatory and housekeeping genes and identify polyadenylation as a novel target for anti-inflammatory drugs.

Introduction and Objectives: Idiopathic Pulmonary Fibrosis (IPF) is a chronic, progressive interstitial lung disease that is refractory to current treatment options. Transforming growth factor (TGF)-β1 is a key pro-fibrotic cytokine that plays a crucial role in IPF pathogenesis. Our group previously demonstrated distinct epigenetic modifications involved in repression of the antifibrotic gene cyclooxygenase-2 (COX-2) in fibroblasts from IPF (F-IPF) lungs compared with fibroblasts from non-fibrotic lungs (F-NL). Epigenetic drugs capable of inhibiting DNA and histone modifications may, therefore, represent a putative novel therapy. The aim of this study was to investigate the ability of 4 epigenetic inhibitors to regulate TGF-β-driven fibrosis in ex vivo mouse lung. Methods: A precision-cut lung slice (PCLS) model of fibrosis was established using the previously described [1] CC10-tTS-rtTA-TGFβ1 transgenic (tgTGF-β1) mouse. The model was first assessed by investigating PCLS overexpression of TGF-β1 in response to stimulation of the transgene by doxycycline treatment. Gene expression of COX-2 and fibrotic markers including collagen were assessed after 4 days of treatment. The anti-fibrotic potential of 4 epigenetic inhibitors; BIX01294 (BIX, inhibitor of G9a histone methyltransferase), 3-deazaneplanocin A (DZNep, inhibitor of EZH2 histone methyltransferase), SAHA (inhibitor of histone deacetylases, HDACs) and Decitabine (DAC, DNA demethylating agent) was investigated. Viability of PCLS was assessed by MTT and Prestoblue® viability assay. Results: Treatment of PCLS from tgTGF-β1 mice with doxycycline induced a concentration-dependent increase in global TGF-β1, pro-fibrotic markers including collagen and pro-inflammatory COX-2, which was comparable to recombinant TGF-β1 treatment. Treatment with three of the epigenetic inhibitors BIX01294, DZNep and DAC did not reduce the pro-fibrotic response following doxycycline treatment. However SAHA demonstrated a significant suppressive effect on COX-2 and collagen expression, while not directly affecting TGF-β1 transgene expression. Conclusions: The data suggests that SAHA has the potential to reduce fibrosis in a TGF-β1 driven model of pulmonary fibrosis. Further work is currently underway to assess the anti-fibrotic potential of this drug in tgTGF-β1 animals.

Airway inflammation is a central feature in the pathophysiology of asthma and cytokine networks play a fundamental role in the chronic inflammatory process. Anti-inflammatory treatment with inhaled corticosteroids provides the mainstay of asthma management in conjunction with bronchodilator therapy. Short-acting β2-adrenoceptor agonists have been used as bronchodilator treatment in asthma for decades and whilst these agents produce useful bronchodilatation through airway smooth muscle relaxation, concerns were raised for several years that they may have deleterious pro-inflammatory effects when given on their own. Short-acting β2-agonists can cause a rebound increase in bronchial responsiveness after cessation of therapy 1 and regular salbutamol use can increase the early response to allergen 2. A number of recent clinical studies, however, particularly with long-acting β2-agonists have contradicted the established dogma that β2-agonists are potentially harmful and may mask underlying inflammation 3–7. Studies with long-acting β2-agonists have shown that when these drugs are given in conjunction with inhaled steroids, they produce beneficial effects on symptoms, airflow and asthma exacerbations 3–7. Whilst these findings may reflect the fact that the best treatment for asthma is to combine an anti-inflammatory drug with a long-acting airway smooth muscle relaxant, they raise the intriguing possibility that there is a beneficial interaction between long-acting β2-adrenoceptor agonists and corticosteroids on part of the inflammatory process. There are a number of in vitro studies which suggest potential anti-inflammatory actions of β2-adrenoceptor agonists which may be complementary or even synergistic with the actions of corticosteroids and the manuscript in this issue by Korn et al . …

Targeted repression of a subset of key genes involved in tissue remodeling is a cardinal feature of idiopathic pulmonary fibrosis (IPF). The mechanism is unclear but is potentially important in disease pathogenesis and therapeutic targeting. We have previously reported that defective histone acetylation is responsible for the repression of the antifibrotic cyclooxygenase-2 gene. Here we extended our study to the repression of another antifibrotic gene, the potent angiostatic chemokine gamma interferon (IFN-gamma)-inducible protein of 10 kDa (IP-10), in lung fibroblasts from patients with IPF. We revealed that this involved not only histone deacetylation, as with cyclooxygenase-2 repression, but also histone H3 hypermethylation, as a result of decreased recruitment of histone acetyltransferases and increased presence of histone deacetylase (HDAC)-containing repressor complexes, histone methyltransferases G9a and SUV39H1, and heterochromatin protein 1 at the IP-10 promoter, leading to reduced transcription factor binding. More importantly, treatment of diseased cells with HDAC or G9a inhibitors similarly reversed the repressive histone deacetylation and hypermethylation and restored IP-10 expression. These findings strongly suggest that epigenetic dysregulation involving interactions between histone deacetylation and hypermethylation is responsible for targeted repression of IP-10 and potentially other antifibrotic genes in fibrotic lung disease and that this is amenable to therapeutic targeting.

Diminished cyclooxygenase 2 (COX-2) expression in fibroblasts, with a resultant defect in the production of the antifibrotic mediator prostaglandin E(2), plays a key role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Here, we have characterized the molecular mechanism. We found that COX-2 mRNA levels in fibroblasts from patients with IPF (F-IPF) were significantly lower than those in fibroblasts from nonfibrotic lungs (F-NL) after transforming growth factor beta1 and interleukin-1beta treatment but that COX-2 mRNA degradation rates were similar, suggesting defective transcription. A reporter gene assay showed that there were no clear differences between F-IPF and F-NL in transcription factor involvement and activation in COX-2 gene transcription. However, a chromatin immunoprecipitation assay revealed that transcription factor binding to the COX-2 promoter in F-IPF was reduced compared to that in F-NL, an effect that was dynamically linked to reduced histone H3 and H4 acetylation due to decreased recruitment of histone acetyltransferases (HATs) and increased recruitment of transcriptional corepressor complexes to the COX-2 promoter. The treatment of F-IPF with histone deacetylase (HDAC) inhibitors together with cytokines increased histone H3 and H4 acetylation. Both HDAC inhibitors and the overexpression of HATs restored cytokine-induced COX-2 mRNA and protein expression in F-IPF. The results demonstrate that epigenetic abnormality in the form of histone hypoacetylation is responsible for diminished COX-2 expression in IPF.