PINX1 and TERT Are Required for TNF-α-Induced Airway Smooth Muscle Chemokine Gene Expression.

Airway smooth muscle (ASM) cells contribute to asthmatic lung pathology with chemokine hypersecretion and increased ASM cell mass. With little recent progress in the development of asthma therapies, a greater understanding of lung inflammation mechanisms has become a priority. Chemokine gene expression in ASM cells is dependent upon NF-κB transcription factor activity. The telomerase/shelterin complex maintains chromosomal telomere ends during cell division. Telomerase is a possible cofactor for NF-κB activity, but its role in NF-κB activity in airway tissue inflammation is not known. In this study, we sought to address two key questions: whether telomerase is involved in inflammation in ASM cells, and whether components of the shelterin complex are also required for an inflammatory response in ASM cells. Telomerase inhibitors and telomerase small interfering RNA (siRNA) reduced TNF-α-induced chemokine expression in ASM cells. Telomerase siRNA and inhibitors reduced NF-κB activity. An siRNA screen of shelterin components identified a requirement for PIN2/TERF1 interacting-telomerase inhibitor 1 (PINX1) in chemokine gene expression. High-level PINX1 overexpression reduced NF-κB reporter activity, but low-level expression amplified NF-κB activity. Coimmunoprecipitation studies showed association of PINX1 and p65. Overexpression of the N terminus (2-252 aa) of PINX1, but not the C-terminal telomerase-inhibitor domain (253-328 aa), amplified TNF-α-induced NF-κB activity. GST pull-downs demonstrated that the N terminus of PINX1 bound more p65 than the C-terminal telomerase-inhibitor domain; these observations were confirmed in whole cells with N-terminal and C-terminal PINX1 immunoprecipitation. We conclude that telomerase and PINX1 are required for chemokine expression in ASM cells and represent significant new targets for future anti-inflammatory therapies for lung diseases, such as asthma.

Interplay between EZH2 and G9a Regulates CXCL10 Gene Repression in Idiopathic Pulmonary Fibrosis.

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-ß1 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.

Role of Rho-Associated Coiled-Coil Forming Kinase Isoforms in Regulation of Stiffness-Induced Myofibroblast Differentiation in Lung Fibrosis.

Fibrosis is a major cause of progressive organ dysfunction in several chronic pulmonary diseases. Rho-associated coiled-coil forming kinase (ROCK) has been shown to be involved in myofibroblast differentiation driven by altered matrix stiffness in a fibrotic state. There are two known ROCK isoforms in humans, ROCK1 and ROCK2, but the specific role of each isoform in myofibroblast differentiation in lung fibrosis remains unknown. To study this, we developed a gelatin methacryloyl hydrogel-based culture system with different stiffness levels relevant to healthy and fibrotic lungs. We have shown that stiff matrix, but not soft matrix, can induce myofibroblast differentiation with high smooth muscle actin isoform (αSMA) expression. Furthermore, our data confirmed that the inhibition of ROCK signaling by a pharmacological inhibitor (i.e., Y27632) attenuates stiffness-induced αSMA expression and fiber assembly in myofibroblasts. To assess the role of ROCK isoforms in this process, we used short interfering RNA to knock down the expression of each isoform. Our data showed that knocking down either ROCK1 or ROCK2 did not result in a reduction in αSMA expression in myofibroblasts on stiff matrix, as opposed to soft matrix, where αSMA expression was reduced significantly. Paradoxically, on stiff matrix, the absence of one isoform (particularly ROCK2) exaggerated αSMA expression and led to thick fiber assembly. Moreover, complete loss of αSMA fiber assembly was seen only in the absence of both ROCK isoforms, suggesting that both isoforms are implicated in this process. Overall, our results indicate the differential role of ROCK isoforms in myofibroblast differentiation on soft and stiff matrices.

Reduced Ets Domain-containing Protein Elk1 Promotes Pulmonary Fibrosis via Increased Integrin αvß6 Expression.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease with high mortality. Active TGFß1 is considered central to the pathogenesis of IPF. A major mechanism of TGFß1 activation in the lung involves the epithelially restricted αvß6 integrin. Expression of the αvß6 integrin is dramatically increased in IPF. How αvß6 integrin expression is regulated in the pulmonary epithelium is unknown. Here we identify a region in the ß6 subunit gene (ITGB6) promoter acting to markedly repress basal gene transcription, which responds to both the Ets domain-containing protein Elk1 (Elk1) and the glucocorticoid receptor (GR). Both Elk1 and GR can regulate αvß6 integrin expression in vitro We demonstrate Elk1 binding to the ITGB6 promoter basally and that manipulation of Elk1 or Elk1 binding alters ITGB6 promoter activity, gene transcription, and αvß6 integrin expression. Crucially, we find that loss of Elk1 causes enhanced Itgb6 expression and exaggerated lung fibrosis in an in vivo model of fibrosis, whereas the GR agonist dexamethasone inhibits Itgb6 expression. Moreover, Elk1 dysregulation is present in epithelium from patients with IPF. These data reveal a novel role for Elk1 regulating ITGB6 expression and highlight how dysregulation of Elk1 can contribute to human disease.

Epigenetic dysregulation of interleukin 8 (CXCL8) hypersecretion in cystic fibrosis airway epithelial cells.

Airway epithelial cells in cystic fibrosis (CF) overexpress Interleukin 8 (CXCL8) through poorly defined mechanisms. CXCL8 transcription is dependent on coordinated binding of CCAAT/enhancer binding protein (C/EBP)ß, nuclear factor (NF)-κB, and activator protein (AP)-1 to the promoter. Here we show abnormal epigenetic regulation is responsible for CXCL8 overexpression in CF cells. Under basal conditions CF cells had increased bromodomain (Brd)3 and Brd4 recruitment and enhanced NF-κB and C/EBPß binding to the CXCL8 promoter compared to non-CF cells due to trimethylation of histone H3 at lysine 4 (H3K4me3) and DNA hypomethylation at CpG6. IL-1ß increased NF-κB, C/EBPß and Brd4 binding. Furthermore, inhibitors of bromodomain and extra-terminal domain family (BET) proteins reduced CXCL8 production in CF cells suggesting a therapeutic target for the BET pathway.

Matrix metalloproteinases -8 and -9 in the Airways, Blood and Urine During Exacerbations of COPD.

Matrix metalloproteinases (MMPs) are elevated in the airways and blood of COPD patients, contributing to disease pathogenesis and tissue remodelling. However, it is not clear if MMP levels in airways, blood and urine are related or if MMP levels are related to disease severity or presence of exacerbations requiring hospitalisation. Seventy-two patients requiring hospitalisation for COPD exacerbations had serum, urine and sputum MMP-8, -9 and active MMP-9 measured by ELISA and gelatin zymography on day one, five and four weeks later (recovery). Clinical history, spirometry, COPD Assessment Test and MRC dyspnoea score were obtained. Twenty-two stable COPD patients had MMP measurements one week apart. During exacerbations, serum and urine MMP-9 were slightly elevated by 17% and 30% compared with recovery values respectively (p = 0.001 and p = 0.026). MMP-8 was not significantly changed. These MMP levels related to serum neutrophil numbers but not to outcome of exacerbations, disease severity measures or smoking status. In clinically stable patients, serum MMP levels did not vary significantly over 7 days, whereas urine MMPs varied by up to nine fold for MMP-8 (p = 0.003). Sputum, serum and urine contained different MMP species and complexes. Median values for sputum active MMP-9 were significantly different from serum (p = 0.035) and urine (p = 0.024). Serum and urine MMPs are only modestly elevated during exacerbations of COPD and unlikely to be useful biomarkers in this clinical setting. Airway, serum and urine MMP levels are independent of each other in COPD patients. Further, MMP levels are variable between patients and do not reflect airflow obstruction.

Human airway smooth muscle cells secrete amphiregulin via bradykinin/COX-2/PGE2, inducing COX-2, CXCL8, and VEGF expression in airway epithelial cells.

Human airway smooth muscle cells (HASMC) contribute to asthma pathophysiology through an increased smooth muscle mass and elevated cytokine/chemokine output. Little is known about how HASMC and the airway epithelium interact to regulate chronic airway inflammation and remodeling. Amphiregulin is a member of the family of epidermal growth factor receptor (EGFR) agonists with cell growth and proinflammatory roles and increased expression in the lungs of asthma patients. Here we show that bradykinin (BK) stimulation of HASMC increases amphiregulin secretion in a mechanism dependent on BK-induced COX-2 expression, increased PGE2 output, and the stimulation of HASMC EP2 and EP4 receptors. Conditioned medium from BK treated HASMC induced CXCL8, VEGF, and COX-2 mRNA and protein accumulation in airway epithelial cells, which were blocked by anti-amphiregulin antibodies and amphiregulin siRNA, suggesting a paracrine effect of HASMC-derived amphiregulin on airway epithelial cells. Consistent with this, recombinant amphiregulin induced CXCL8, VEGF, and COX-2 in airway epithelial cells. Finally, we found that conditioned media from amphiregulin-stimulated airway epithelial cells induced amphiregulin expression in HASMC and that this was dependent on airway epithelial cell COX-2 activity. Our study provides evidence of a dynamic axis of interaction between HASMC and epithelial cells that amplifies CXCL8, VEGF, COX-2, and amphiregulin production.

CXCL8 histone H3 acetylation is dysfunctional in airway smooth muscle in asthma: regulation by BET.

Asthma is characterized by airway inflammation and remodeling and CXCL8 is a CXC chemokine that drives steroid-resistant neutrophilic airway inflammation. We have shown that airway smooth muscle (ASM) cells isolated from asthmatic individuals secrete more CXCL8 than cells from nonasthmatic individuals. Here we investigated chromatin modifications at the CXCL8 promoter in ASM cells from nonasthmatic and asthmatic donors to further understand how CXCL8 is dysregulated in asthma. ASM cells from asthmatic donors had increased histone H3 acetylation, specifically histone H3K18 acetylation, and increased binding of histone acetyltransferase p300 compared with nonasthmatic donors but no differences in CXCL8 DNA methylation. The acetylation reader proteins Brd3 and Brd4 were bound to the CXCL8 promoter and Brd inhibitors inhibited CXCL8 secretion from ASM cells by disrupting Brd4 and RNA polymerase II binding to the CXCL8 promoter. Our results show a novel dysregulation of CXCL8 transcriptional regulation in asthma characterized by a promoter complex that is abnormal in ASM cells isolated from asthmatic donors and can be modulated by Brd inhibitors. Brd inhibitors may provide a new therapeutic strategy for steroid-resistant inflammation.

Pseudomonas aeruginosa quorum sensing molecules correlate with clinical status in cystic fibrosis.

Pseudomonas aeruginosa produces quorum sensing signal molecules that are potential biomarkers for infection.A prospective study of 60 cystic fibrosis patients with chronic P. aeruginosa, who required intravenous antibiotics for pulmonary exacerbations, was undertaken. Clinical measurements and biological samples were obtained at the start and end of the treatment period. Additional data were available for 29 of these patients when they were clinically stable.Cross-sectionally, quorum sensing signal molecules were detectable in the sputum, plasma and urine of 86%, 75% and 83% patients, respectively. They were positively correlated between the three biofluids. Positive correlations were observed for most quorum sensing signal molecules in sputum, plasma and urine, with quantitative measures of pulmonary P. aeruginosa load at the start of a pulmonary exacerbation. Plasma concentrations of 2-nonyl-4-hydroxy-quinoline (NHQ) were significantly higher at the start of a pulmonary exacerbation compared to clinical stability (p<0.01). Following the administration of systemic antibiotics, plasma 2-heptyl-4-hydroxyquinoline (p=0.02) and NHQ concentrations (p<0.01) decreased significantly.In conclusion, quorum sensing signal molecules are detectable in cystic fibrosis patients with pulmonary P. aeruginosa infection and are positively correlated with quantitative measures of P. aeruginosa. NHQ correlates with clinical status and has potential as a novel biomarker for P. aeruginosa infection.

Clinical and inflammatory characteristics of the European U-BIOPRED adult severe asthma cohort.

U-BIOPRED is a European Union consortium of 20 academic institutions, 11 pharmaceutical companies and six patient organisations with the objective of improving the understanding of asthma disease mechanisms using a systems biology approach.This cross-sectional assessment of adults with severe asthma, mild/moderate asthma and healthy controls from 11 European countries consisted of analyses of patient-reported outcomes, lung function, blood and airway inflammatory measurements.Patients with severe asthma (nonsmokers, n=311; smokers/ex-smokers, n=110) had more symptoms and exacerbations compared to patients with mild/moderate disease (n=88) (2.5 exacerbations versus 0.4 in the preceding 12 months; p<0.001), with worse quality of life, and higher levels of anxiety and depression. They also had a higher incidence of nasal polyps and gastro-oesophageal reflux with lower lung function. Sputum eosinophil count was higher in severe asthma compared to mild/moderate asthma (median count 2.99% versus 1.05%; p=0.004) despite treatment with higher doses of inhaled and/or oral corticosteroids.Consistent with other severe asthma cohorts, U-BIOPRED is characterised by poor symptom control, increased comorbidity and airway inflammation, despite high levels of treatment. It is well suited to identify asthma phenotypes using the array of "omic" datasets that are at the core of this systems medicine approach.

Accelerated extracellular matrix turnover during exacerbations of COPD.

BACKGROUND: Exacerbations of chronic obstructive pulmonary disease (COPD) contribute significantly to disease progression. However, the effect on tissue structure and turnover is not well described. There is an urgent clinical need for biomarkers of disease activity associated with disease progression. Extracellular matrix (ECM) turnover reflects activity in tissues and consequently assessment of ECM turnover may serve as biomarkers of disease activity. We hypothesized that the turnover of lung ECM proteins were altered during exacerbations of COPD. METHODS: 69 patients with COPD hospitalised for an exacerbation were recruited at admission and returned for a 4 weeks follow-up. Competitive ELISAs measuring circulating protein fragments in serum or plasma assessed the formation and degradation of collagen types III (Pro-C3 and C3M, respectively), IV (P4NP 7S and C4M, respectively), and VI (Pro-C6 and C6M, respectively), and degradation of elastin (ELM7 and EL-NE) and versican (VCANM). RESULTS: Circulating levels of C3M, C4M, C6M, ELM7, and EL-NE were elevated during an exacerbation of COPD as compared to follow-up (all P <0.0001), while VCANM levels were decreased (P <0.0001). Pro-C6 levels were decreased and P4NP 7S levels were elevated during exacerbation (P <0.0001). Pro-C3 levels were unchanged. At time of exacerbation, degradation/formation ratios were increased for collagen types III and VI and decreased for collagen type IV. CONCLUSIONS: Exacerbations of COPD resulted in elevated levels of circulating fragments of structural proteins, which may serve as markers of disease activity. This suggests that patients with COPD have accelerated ECM turnover during exacerbations which may be related to disease progression.

A central role for G9a and EZH2 in the epigenetic silencing of cyclooxygenase-2 in idiopathic pulmonary fibrosis.

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.

Inhibition of polyadenylation reduces inflammatory gene induction.

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.

Abnormal histone methylation is responsible for increased vascular endothelial growth factor 165a secretion from airway smooth muscle cells in asthma.

Vascular endothelial growth factor (VEGF), a key angiogenic molecule, is aberrantly expressed in several diseases including asthma where it contributes to bronchial vascular remodeling and chronic inflammation. Asthmatic human airway smooth muscle cells hypersecrete VEGF, but the mechanism is unclear. In this study, we defined the mechanism in human airway smooth muscle cells from nonasthmatic and asthmatic patients. We found that asthmatic cells lacked a repression complex at the VEGF promoter, which was present in nonasthmatic cells. Recruitment of G9A, trimethylation of histone H3 at lysine 9 (H3K9me3), and a resultant decrease in RNA polymerase II at the VEGF promoter was critical to repression of VEGF secretion in nonasthmatic cells. At the asthmatic promoter, H3K9me3 was absent because of failed recruitment of G9a; RNA polymerase II binding, in association with TATA-binding protein-associated factor 1, was increased; H3K4me3 was present; and Sp1 binding was exaggerated and sustained. In contrast, DNA methylation and histone acetylation were similar in asthmatic and nonasthmatic cells. This is the first study, to our knowledge, to show that airway cells in asthma have altered epigenetic regulation of remodeling gene(s). Histone methylation at genes such as VEGF may be an important new therapeutic target.

Elevated SP-1 transcription factor expression and activity drives basal and hypoxia-induced vascular endothelial growth factor (VEGF) expression in non-small cell lung cancer.

VEGF plays a central role in angiogenesis in cancer. Non-small cell lung cancer (NSCLC) tumors have increased microvascular density, localized hypoxia, and high VEGF expression levels; however, there is a lack of understanding of how oncogenic and tumor microenvironment changes such as hypoxia lead to greater VEGF expression in lung and other cancers. We show that NSCLC cells secreted higher levels of VEGF than normal airway epithelial cells. Actinomycin D inhibited all NSCLC VEGF secretion, and VEGF minimal promoter-luciferase reporter constructs were constitutively active until the last 85 base pairs before the transcription start site containing three SP-1 transcription factor-binding sites; mutation of these VEGF promoter SP-1-binding sites eliminated VEGF promoter activity. Furthermore, dominant negative SP-1, mithramycin A, and SP-1 shRNA decreased VEGF promoter activity, whereas overexpression of SP-1 increased VEGF promoter activity. Chromatin immunoprecipitation assays demonstrated SP-1, p300, and PCA/F histone acetyltransferase binding and histone H4 hyperacetylation at the VEGF promoter in NSCLC cells. Cultured NSCLC cells expressed higher levels of SP-1 protein than normal airway epithelial cells, and double-fluorescence immunohistochemistry showed a strong correlation between SP-1 and VEGF in human NSCLC tumors. In addition, hypoxia-driven VEGF expression in NSCLC cells was SP-1-dependent, with hypoxia increasing SP-1 activity and binding to the VEGF promoter. These studies are the first to demonstrate that overexpression of SP-1 plays a central role in hypoxia-induced VEGF secretion.

Integrin αvß5-mediated TGF-ß activation by airway smooth muscle cells in asthma.

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-ß 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-ß is synthesized as a latent complex, sequestered in the extracellular matrix, and requires activation for functionality. Activation of latent TGF-ß is the rate-limiting step in its bioavailability. This study investigated the effect of the contraction agonists LPA and methacholine on TGF-ß activation by HASM cells and its role in the development of asthmatic airway remodeling. The data presented show that LPA and methacholine induced TGF-ß activation by HASM cells via the integrin αvß5. Our findings highlight the importance of the ß5 cytoplasmic domain because a polymorphism in the ß5 subunit rendered the integrin unable to activate TGF-ß. To our knowledge, this is the first description of a biologically relevant integrin that is unable to activate TGF-ß. These data demonstrate that murine airway smooth muscle cells express αvß5 integrins and activate TGF-ß. Finally, these data show that inhibition, or genetic loss, of αvß5 reduces allergen-induced increases in airway smooth muscle thickness in two models of asthma. These data highlight a mechanism of TGF-ß activation in asthma and support the hypothesis that bronchoconstriction promotes airway remodeling via integrin mediated TGF-ß activation.

Future bronchodilator therapy: a bitter pill to swallow?

Maintenance of airway tone, prevention of airway obstruction, and acute relief from bronchospasm are key targets of asthma therapy. This role is currently performed by ß-agonists. However, chronic use of ß-agonists to treat asthma is associated with desensitization of ß-agonist signaling and a resultant loss of bronchodilator effect, worsening of airway hyperreactivity, and increased incidence of asthma-related morbidity and mortality. There have been several attempts to identify novel non-ß-agonist bronchodilators including ATP-sensitive potassium channel (K(ATP)) agonists such as cromakalim and its active enantiomer BRL-38227 and the cGMP activators atrial natriuretic peptide (ANP) and BAY 41-22722. However, these either have not made it to clinical trial, required high doses, had little effect in patients, or had a high incidence of side effects. Recent data suggests that a novel bronchodilator target exists, the bitter taste receptor TAS2R. Two recent studies [An SS, Wang WC, Koziol-White CJ, Ahn K, Lee DY, Kurten RC, Panettieri RA Jr, Liggett SB. Am J Physiol Lung Cell Mol Physiol 303: L304-L311, 2012; Pulkkinen V, Manson ML, Säfholm J, Adner M, Dahlén SE. Am J Physiol Lung Cell Mol Physiol. doi:10.1152/ajplung.00205.2012.] provide new understanding of the signaling pathways utilized by TAS2Rs to mediate their bronchodilatory effects and how TAS2R-mediated bronchodilation is affected by ß-agonist signaling desensitization. As our understanding of TAS2Rs and their agonists increases, they move closer to a viable therapeutic option; however, further definition is still required and questions remain to be answered. This editorial focus discusses these studies within the context of existing literature and raises questions and challenges for the future development of bitter (better?) therapies for asthma.

Ciclesonide inhibits TNFα- and IL-1ß-induced monocyte chemotactic protein-1 (MCP-1/CCL2) secretion from human airway smooth muscle cells.

Monocyte chemotactic protein-1 (MCP-1) is a member of the CC family of cytokines. It has monocyte and lymphocyte chemotactic activity and stimulates histamine release from basophils. MCP-1 is implicated in the pathogenesis of inflammatory diseases, including asthma. The airway smooth muscle (ASM) layer is thickened in asthma, and the growth factors and cytokines secreted by ASM cells play a role in the inflammatory response of the bronchial wall. Glucocorticoids and ß(2)-agonists are first-line drug treatments for asthma. Little is known about the effect of asthma treatments on MCP-1 production from human ASM cells. Here, we determined the effect of ciclesonide (a glucocorticoid) and formoterol (a ß(2)-agonist) on MCP-1 production from human ASM cells. TNFα and IL-1ß induced MCP-1 secretion from human ASM cells. Formoterol had no effect on MCP-1 expression, while ciclesonide significantly inhibited IL-1ß- and TNFα-induced MCP-1. Furthermore, ciclesonide inhibited IL-1ß- and TNFα-induced MCP-1 mRNA and IL-1ß- and TNFα-induced MCP-1 promoter and enhancer luciferase reporters. Western blots showed that ciclesonide had no effect on IκB degradation. Finally, ciclesonide inhibited an NF-κB luciferase reporter. Our data show that ciclesonide inhibits IL-1ß- and TNFα-induced MCP-1 production from human ASM cells via a transcriptional mechanism involving inhibition of NF-κB binding.

Expression of prostaglandin E synthases in periodontitis immunolocalization and cellular regulation.

The inflammatory mediator prostaglandin E(2) (PGE(2)) is implicated in the pathogenesis of chronic inflammatory diseases including periodontitis; it is synthesized by cyclooxygenases (COX) and the prostaglandin E synthases mPGES-1, mPGES-2, and cPGES. The distribution of PGES in gingival tissue of patients with periodontitis and the contribution of these enzymes to inflammation-induced PGE(2) synthesis in different cell types was investigated. In gingival biopsies, positive staining for PGES was observed in fibroblasts and endothelial, smooth muscle, epithelial, and immune cells. To further explore the contribution of PGES to inflammation-induced PGE(2) production, in vitro cell culture experiments were performed using fibroblasts and endothelial, smooth muscle, and mast cells. All cell types expressed PGES and COX-2, resulting in basal levels of PGE(2) synthesis. In response to tumor necrosis factor (TNF-α), IL-1ß, and cocultured lymphocytes, however, mPGES-1 and COX-2 protein expression increased in fibroblasts and smooth muscle cells, accompanied by increased PGE(2), whereas mPGES-2 and cPGES were unaffected. In endothelial cells, TNF-α increased PGE(2) production only via COX-2 expression, whereas in mast cells the cytokines did not affect PGE(2) enzyme expression or PGE(2) production. Furthermore, PGE(2) production was diminished in gingival fibroblasts derived from mPGES-1 knockout mice, compared with wild-type fibroblasts. These results suggest that fibroblasts and smooth muscle cells are important sources of mPGES-1, which may contribute to increased PGE(2) production in the inflammatory condition periodontitis.

Endothelin-1 (ET-1) increases the expression of remodeling genes in vascular smooth muscle through linked calcium and cAMP pathways: role of a phospholipase A(2)(cPLA(2))/cyclooxygenase-2 (COX-2)/prostacyclin receptor-dependent autocrine loop.

Several important genes that are involved in inflammation and tissue remodeling are switched on by virtue of CRE response elements in their promoters. The upstream signaling mechanisms that inflammatory mediators use to activate cAMP response elements (CREs) are poorly understood. Endothelin (ET) is an important vasoactive mediator that plays roles in inflammation, vascular remodeling, angiogenesis, and carcinogenesis by activating 7 transmembrane G protein-coupled receptors (GPCR). Here we characterized the mechanisms ET-1 uses to regulate CRE-dependent remodeling genes in pulmonary vascular smooth muscle cells. These studies revealed activation pathways involving a cyclooxygenase-2 (COX-2)/prostacyclin receptor (IP receptor) autocrine loop and an interlinked calcium-dependent pathway. We found that ET-1 activated several CRE response genes in vascular smooth muscle cells, particularly COX-2, amphiregulin, follistatin, inhibin-beta-A, and CYR61. ET-1 also activated two other genes epiregulin and HB-EGF. Amphiregulin, follistatin, and inhibin-beta-A and epiregulin were activated by an autocrine loop involving cPLA2, arachidonic acid release, COX-2-dependent PGI(2) synthesis, and IP receptor-linked elevation of cAMP leading to CRE transcription activation. In contrast COX-2, CYR61, and HB-EGF transcription were regulated in a calcium-dependent, COX-2 independent, manner. Observations with IP receptor antagonists and COX-2 inhibitors were confirmed with IP receptor or COX-2-specific small interfering RNAs. ET-1 increases in intracellular calcium and gene transcription were dependent upon ET(a) activation and calcium influx through T type voltage-dependent calcium channels. These studies give important insights into the upstream signaling mechanisms used by G protein-coupled receptor-linked mediators such as ET-1, to activate CRE response genes involved in angiogenesis, vascular remodeling, inflammation, and carcinogenesis.