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Tristetraprolin (TTP) is a destabilizing mRNA binding protein known to regulate gene expression of a wide variety of targets, including those that control inflammation. TTP expression, regulation and function is controlled by phosphorylation. While the importance of key serine (S) sites (S52 and S178 in mice and S186 in humans) has been recognized, other sites on the hyperphosphorylated TTP protein have more recently emerged as playing an important role in regulating cellular signalling and downstream functions of TTP. In order to propel investigation of TTP and fully exploit its potential as a drug target in inflammatory disease, this review will catalogue TTP phosphorylation sites in both the murine and human TTP protein, the known and unknown roles and functions of these sites, the kinases and phosphatases that act upon TTP and overview methodological approaches to increase our knowledge of this important protein regulated by phosphorylation.

The role of the ATP binding cassette transporter A1 (ABCA1) in maintaining cellular lipid homeostasis in cardiovascular disease is well established. More recently, the important beneficial role played by ABCA1 in modulating pathogenic disease mechanisms, such as inflammation, in a broad range of chronic conditions has been realised. These studies position ABCA1 as a potential therapeutic target in a diverse range of diseases where inflammation is an underlying cause. Chronic respiratory conditions such as asthma and chronic obstructive pulmonary disease (COPD) are driven by inflammation, and as such, there is now a growing recognition that we need a greater understanding of the signaling pathways responsible for regulation of ABCA1 expression in this clinical context. While the signaling pathways responsible for cholesterol-mediated ABCA1 expression have been clearly delineated through decades of studies in the atherosclerosis field, and thus far appear to be translatable to the respiratory field, less is known about the cholesterol-independent signaling pathways that can modulate ABCA1 expression in inflammatory lung disease. This review will identify the various signaling pathways and ligands that are associated with the regulation of ABCA1 expression and may be exploited in future as therapeutic targets in the setting of chronic inflammatory lung diseases.

Highlights • TTP negatively regulates expression of large numbers of inflammatory mediators. • A TTP knock-out mouse suffers chronic and severe inflammatory pathology. • TTP is multiply phosphorylated. Phosphorylation of serines 52 and 178 is mediated by the MAPK p38-activated kinase MK2. • Phosphorylation of TTP at serines 52 and 178 causes its inactivation, and is permissive for expression of TTP-regulated inflammatory mediators. • Inflammation is reduced by genetically or pharmaceutically interfering with the phosphorylation of serines 52 and 178., Tristetraprolin (TTP) is an RNA-destabilizing protein that exerts profound anti-inflammatory effects by inhibiting the expression of tumour necrosis factor and many other inflammatory mediators. The mitogen-activated protein kinase (MAPK) p38 signaling pathway controls the strength and duration of inflammatory responses by regulating both the expression and function of TTP. The kinase MK2 (MAPK activated kinase 2) is activated by MAPK p38, and in turn phosphorylates TTP at two critical serine residues. One consequence of these phosphorylations is the protection of TTP from proteasome-mediated degradation. Another consequence is the loss of mRNA destabilizing activity. The control of TTP expression and function by the MAPK p38 pathway provides an elegant mechanism for coupling the on and off phases of inflammatory responses, and dictating the precise kinetics of expression of individual inflammatory mediators.

© 2019 Institute of Pharmacology, Polish Academy of Sciences Background: The pathogenesis of chronic obstructive pulmonary disease (COPD) is associated with dyslipidemia, an established co-morbidity. Statins treat hypercholesterolemia, but more recently have been trailed in the setting of COPD for their potential anti-inflammatory benefits. The outcomes of prospective trials however have been inconsistent. Thus, we hypothesize that the variation in results may have been due to statin-induced downregulation of ATP-binding cassette transporter A1 (ABCA1), thereby reducing cholesterol export. This study aims to elucidate whether statin treatment in a cellular model of COPD leads to a decrease in ABCA1 protein expression. Methods: To mimic the inflammatory environment of COPD, two commonly used lung epithelial cell lines (BEAS-2B and A549) were treated with tumor necrosis factor (TNF), and co-treated with cholesterol/25-hydroxycholesterol (25-OH) to mimic dyslipidemia. ABCA1 protein was detected by Western Blotting. Results: We unexpectedly showed that statins did not affect ABCA1 expression. However, the LXR agonist T0901317 significantly increased ABCA1 expression in both cell lines, while TNF, cholesterol or 25-OH induced ABCA1 protein upregulation in BEAS-2B cells, indicating cell line differences in response. There was also evidence of synergistic impacts of combined treatments on ABCA1 upregulation in BEAS-2B cells. Conclusion: Statins did not have an impact on ABCA1 expression in lung epithelial cell lines, disproving our original hypothesis. However, we showed for the first time, the effect of the inflammatory cytokine TNF, cholesterol/25-OH, statins and the LXR agonist T0901317 on expression of ABCA1 transporter protein in human lung epithelial cell lines in vitro. We hope that these in vitro studies may prove beneficial for addressing dyslipidemia in COPD in the future.

© 2018 Wiley Periodicals, Inc. Prostaglandin E 2 (PGE 2 ) is a key prostanoid known to have both proinflammatory and anti-inflammatory impact in the context of chronic respiratory diseases. We hypothesize that these opposing effects may be the result of different prostanoid E (EP) receptor-mediated signaling pathways. In this study, we focus on two of the four EP receptors, EP 2 and EP 4 , as they are known to induce cyclic adenosine monophosphate (cAMP)-dependent signaling pathways. Using primary human airway smooth muscle (ASM) cells, we first focussed on the PGE 2 -induced production of two cAMP-dependent proinflammatory mediators: interleukin 6 (IL-6) and cyclo-oxygenase 2 production. We show that PGE 2 -induced IL-6 protein secretion occurs via an EP 2 -mediated pathway, in a manner independent of receptor-mediated effects on messenger RNA (mRNA) expression and temporal activation kinetics of the transcription factor cAMP response element binding. Moreover, stimulation of ASM with PGE 2 did not establish a positive, receptor-mediated, feedback loop, as mRNA expression for EP 2 and EP 4 receptors were not upregulated and receptor antagonists were without effect. Our studies revealed that the EP 2 , but not the EP 4 , receptor is responsible for β 2 -adrenergic desensitization induced by PGE 2 . We demonstrate that PGE 2 -induced heterologous receptor desensitization responsible for tachyphylaxis to short- (salbutamol) or long- (formoterol) β 2 -agonists (measured by cAMP release) can be reversed by the EP 2 receptor antagonist PF-04418948. Importantly, this study highlights that inhibiting the EP 2 receptor restores β 2 -adrenergic receptor function in vitro and offers an attractive novel therapeutic target for treating infectious exacerbations in people suffering from chronic respiratory diseases in the future.

© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society Chronic respiratory diseases are among the leading causes of mortality worldwide, with the major contributor, chronic obstructive pulmonary disease (COPD) accounting for approximately 3 million deaths annually. Frequent acute exacerbations (AEs) of COPD (AECOPD) drive clinical and functional decline in COPD and are associated with accelerated loss of lung function, increased mortality, decreased health-related quality of life and significant economic costs. Infections with a small subgroup of pathogens precipitate the majority of AEs and consequently constitute a significant comorbidity in COPD. However, current pharmacological interventions are ineffective in preventing infectious exacerbations and their treatment is compromised by the rapid development of antibiotic resistance. Thus, alternative preventative therapies need to be considered. Pathogen adherence to the pulmonary epithelium through host receptors is the prerequisite step for invasion and subsequent infection of surrounding structures. Thus, disruption of bacterial-host cell interactions with receptor antagonists or modulation of the ensuing inflammatory profile present attractive avenues for therapeutic development. This review explores key mediators of pathogen-host interactions that may offer new therapeutic targets with the potential to prevent viral/bacterial-mediated AECOPD. There are several conceptual and methodological hurdles hampering the development of new therapies that require further research and resolution.

© 2019, Springer Nature Switzerland AG. Tristetraprolin (TTP) is an anti-inflammatory molecule known to post-transcriptionally regulate cytokine production and is, therefore, an attractive drug target for chronic respiratory diseases driven by inflammation, such as asthma and chronic obstructive pulmonary disease. Our recent in vitro studies in primary human airway smooth (ASM) cells have confirmed the essential anti-inflammatory role played by TTP as a critical partner in a cytokine regulatory network. However, several unanswered questions remain. While prior in vitro studies have suggested that TTP is regulated in a cAMP-mediated manner, raising the possibility that this may be one of the ways in which β2-agonists achieve beneficial effects beyond bronchodilation, the impact of β2-agonists on ASM cells is unknown. Furthermore, the effect of prostaglandin E2 (PGE2) on TTP expression in ASM cells has not been reported. We address this herein and reveal, for the first time, that TTP is not regulated by cAMP-activating agents nor following treatment with long-acting β2-agonists. However, PGE2 does induce TTP mRNA expression and protein upregulation in ASM cells. Although the underlying mechanism of action remains undefined, we can confirm that PGE2-induced TTP upregulation is not mediated via cAMP, or EP2/EP4 receptor activation, and occurred in a manner independent of the p38 MAPK-mediated pathway. Taken together, these data confirm that β2-agonists do not upregulate TTP in human ASM cells and indicate that another way in which PGE2 may achieve beneficial effects in asthma and COPD may be via upregulation of the master controller of inflammation—TTP.

Copyright © 2019 Hoppstädter and Ammit. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Glucocorticoids (GCs) potently inhibit pro-inflammatory responses and are widely used for the treatment of inflammatory diseases, such as allergies, autoimmune disorders, and asthma. Dual-specificity phosphatase 1 (DUSP1), also known as mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1), exerts its effects by dephosphorylation of MAPKs, i.e., extracellular-signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK). Endogenous DUSP1 expression is tightly regulated at multiple levels, involving both transcriptional and post-transcriptional mechanisms. DUSP1 has emerged as a central mediator in the resolution of inflammation, and upregulation of DUSP1 by GCs has been suggested to be a key mechanism of GC actions. In this review, we discuss the impact of DUSP1 on the efficacy of GC-mediated suppression of inflammation and address the underlying mechanisms.

© 2018 Elsevier B.V. Despite many therapeutic advancements over the past decade, the continued rise in chronic inflammatory lung diseases incidence has driven the need to identify and develop new therapeutic strategies, with superior efficacy to treat these diseases. Statins are one class of drug that could potentially be repurposed as an alternative treatment for chronic lung diseases. They are currently used to treat hypercholesterolemia by inhibiting the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, that catalyses the rate limiting step in the mevalonate biosynthesis pathway, a key intermediate in cholesterol metabolism. Recent research has identified statins to have other protective pleiotropic properties including anti-inflammatory, anti-oxidant, muco-inhibitory effects that may be beneficial for the treatment of chronic inflammatory lung diseases. However, clinical studies have yielded conflicting results. This review will summarise some of the current evidences for statins pleiotropic effects that could be applied for the treatment of chronic inflammatory lung diseases, their mechanisms of actions, and the potential to repurpose statins as an inhaled therapy, including a detailed discussion on their different physical-chemical properties and how these characteristics could ultimately affect treatment efficacies. The repurposing of statins from conventional anti-cholesterol oral therapy to inhaled anti-inflammatory formulation is promising, as it provides direct delivery to the airways, reduced risk of side effects, increased bioavailability and tailored physical-chemical properties for enhanced efficacy.

© 2018 Elsevier Ltd Arachidonic acid is metabolized by cyclooxygenases (COX-1 and COX-2) into various prostanoids which exert different functions in mammalian physiology. One of these prostanoids, prostaglandin E 2 (PGE 2 ), interacts with four different G protein-coupled receptors, named EP 1 , EP 2 , EP 3 and EP 4 , to initiate different downstream signalling pathways. Prostanoid receptors are diversely expressed throughout different tissues all over the body and PGE 2 is responsible for a large variety of beneficial and disadvantageous effects. We have recently achieved a greater understanding of the biology of prostanoid E receptors and the potential for specific drug targeting with the advent of potent and selective EP receptor agonists and antagonists. This has important implications for lung health and disease as PGE 2 -mediated EP receptor activation impacts upon migration of airway smooth muscle cells, airway microvascular leak, tone regulation of pulmonary blood vessels, mast cell degranulation, bronchodilatation, cough, angiogenesis and airway inflammation, to name a few. In this review, we overview the EP receptor family and the related signalling pathways, summarize a variety of EP 1-4 receptor agonists and antagonists, provide an overview of pharmacological tools used to implicate EP receptor function in the context of respiratory health and disease and finally highlight some of the more selective pharmacological reagents that have recently been developed. The availability of selective pharmacological agonists and antagonists for the distinct EP receptors, as well as the development of specific prostanoid receptor knock-out mice, offer hitherto unattainable opportunities for achieving an in depth understanding of the role and function of PGE 2 in respiratory disease and the exciting potential of targeting EP receptors more broadly.

© 2017 Novel para-sulfonatocalix[4]arene (sCX[4]) and polyamidoamine (PAMAM) dendrimer nanocomplexes were evaluated as delivery vehicles for the platinum anticancer agent [(1,10-phenanthroline)(1S,2S-diaminocyclohexane)platinum(II)] chloride (PHENSS). Different ratios of sCX[4] to PHENSS were tested for their compatibility, with a ratio of 6:1 sCX[4]:PHENSS having the best solubility. The loading of sCX[4], and sCX[4]-bound PHENSS, onto three different generations of PAMAM dendrimers (G3.0–5.0) was examined using UV–visible spectrophotometry. The quantity of sCX[4] bound was found to increase exponentially with dendrimer size: G3, 15 sCX[4] molecules per dendrimer; G4, 37; and G5, 78. Similarly, the loading of sCX[4]-bound PHENSS also increased with increasing dendrimer size: G3, 7 PHENSS molecules per dendrimer; G4, 14; and G5, 28.5. The loading of sCX[4]-bound PHENSS molecules is significantly lower when compared with that of sCX[4], which indicates that less than half of the binding sites were occupied (45, 44, and 44%, respectively). By 1H NMR and UV–vis analysis, the nanocomplex was found to be stable in NaCl solutions at concentrations up to 150 mM. While PHENSS is more active in vitro than cisplatin against the human breast cancer cell line, MCF-7, delivery of PHENSS using the sCX[4]-dendrimer nanocomplexes, regardless of dendrimer generation, had little effect on PHENSS cytotoxicity. The results of this study may have application in the delivery of a variety of small molecule metal-based drugs for which chemical conjugation to a nanoparticle is undesired or not feasible.

© 2017 The xanthine doxofylline has been examined in clinical trials and shown to have efficacy and greater tolerability than theophylline in asthma and chronic obstructive pulmonary disease. The ‘novofylline’ doxofylline has demonstrated bronchodilatory and anti-inflammatory actions in in vivo and ex vivo experimental models of respiratory disease. However, there are limited studies in vitro. We address this herein and examine whether doxofylline has anti-inflammatory impact on primary cultures of airway smooth muscle (ASM) cells. We conduct a series of investigations comparing and contrasting doxofylline with the archetypal xanthine, theophylline, and the specific phosphodiesterase (PDE) 4 inhibitor, cilomilast. We confirm that the xanthine drugs do not have action as PDE inhibitors in ASM cells. Unlike cilomilast, doxofylline (and theophylline) do not increase cAMP production in ASM cells induced by long-acting β2-agonist formoterol. Similar to theophylline, and consistent with the lack of cAMP potentiation, doxofylline does not augment formoterol-induced upregulation of the anti-inflammatory protein mitogen-activated protein kinase phosphatase 1 (MKP-1). However, when we examine the effect of doxofylline on secretion of the interleukin 8 from ASM cells stimulated by tumour necrosis factor (an in vitro surrogate measure of inflammation), there was no repression of inflammation. This is in contrast to the anti-inflammatory impact exerted by theophylline and cilomilast in confirmatory experiments. In summary, our study is the first to examine the effect of doxofylline on ASM cells in vitro and highlights some distinct differences between two key members of xanthine drug family, doxofylline and theophylline.

© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Mitogen-activated protein kinase phosphatases (MKPs) are over-expressed in many cancers. The increased levels of MKP enzymes protect cells from apoptosis induced by anticancer drugs, and thereby decrease the efficacy of the drug. Consequently, there is considerable interest in the development of agents that can down-regulate the production of the MKP enzymes or inhibit their catalytic activities. We have examined the ability of a series of oligonuclear polypyridylruthenium(II) complexes to inhibit the activity of MKP-1 and MKP-3. The results demonstrated that two tetranuclear complexes inhibit the activity of MKP-1 and MKP-3 at 10-20 μM concentrations. The ability of the ruthenium complexes to inhibit the production of MKP-1 in live cancer cells was demonstrated through Western blotting assays, while real-time reverse transcription polymerase chain reaction assays demonstrated the tetranuclear complexes decreased the amount of mkp-1 mRNA produced in cancer cells. The results of this study suggest that tetranuclear ruthenium complexes could be used to enhance the antiproliferative effect of anticancer drugs.

©2016 Published by Elsevier B.V. Prostaglandin E2 (PGE2) is a prostanoid with diverse actions in health and disease. In chronic respiratory diseases driven by inflammation, PGE2 has both positive and negative effects. An enhanced understanding of the receptor-mediated cellular signalling pathways induced by PGE2 may help us separate the beneficial properties from unwanted actions of this important prostaglandin. PGE2 is known to exert anti-inflammatory and bronchoprotective actions in human airways. To date however, whether PGE2 increases production of the anti-inflammatory protein MAPK phosphatase 1 (MKP-1) was unknown. We address this herein and use primary cultures of human airway smooth muscle (ASM) cells to show that PGE2 increases MKP-1 mRNA and protein upregulation in a concentration-dependent manner. We explore the signalling pathways responsible and show that PGE2-induces CREB phosphorylation, not p38 MAPK activation, in ASM cells. Moreover, we utilize selective antagonists of EP2 (PF-04418948) and EP4 receptors (GW 627368X) to begin to identify EP-mediated functional outcomes in ASM cells in vitro. Taken together with earlier studies, our data suggest that PGE2 increases production of the anti-inflammatory protein MKP-1 via cAMP/CREB-mediated cellular signalling in ASM cells and demonstrates that EP2 may, in part, be involved.

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. Background IL-17A plays an important role in respiratory disease and is a known regulator of pulmonary inflammation and immunity. Recent studies have linked IL-17A with exacerbation in asthma and COPD. We have shown that the enzyme cyclooxygenase-2 (COX-2) and its prostanoid products, prostaglandin E2 (PGE2) in particular, are key contributors in in vitro models of infectious exacerbation; however, the impact of IL-17A was not known. Methods and Results We address this herein and show that IL-17A induces a robust and sustained upregulation of COX-2 protein and PGE2 secretion from airway smooth muscle (ASM) cells. COX-2 can be regulated at transcriptional, post-transcriptional and/or post-translational levels. We have elucidated the underlying molecular mechanisms responsible for the sustained upregulation of TNF-α-induced COX-2 by IL-17A in ASM cells and show that is not via increased COX-2 gene expression. Instead, TNF-α-induced COX-2 upregulation is subject to regulation by the proteasome, and IL-17A acts to increase TNF-α-induced COX-2 protein stability as confirmed by cycloheximide chase experiments. In this way, IL-17A acts to amplify the COX-2-mediated effects of TNF-α and greatly enhances PGE2 secretion from ASM cells. Conclusion As PGE2 is a multifunctional prostanoid with diverse roles in respiratory disease, our studies demonstrate a novel function for IL-17A in airway inflammation by showing for the first time that IL-17A impacts on the COX-2/PGE2 pathway, molecules known to contribute to disease exacerbation.

© 2016 Wiley Periodicals, Inc. Exaggerated cytokine secretion drives pathogenesis of a number of chronic inflammatory diseases, including asthma. Anti-inflammatory pharmacotherapies, including corticosteroids, are front-line therapies and although they have proven clinical utility, the molecular mechanisms responsible for their actions are not fully understood. The corticosteroid-inducible gene, mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP-1, DUSP1) has emerged as a key molecule responsible for the repressive effects of steroids. MKP-1 is known to deactivate p38 MAPK phosphorylation and can control the expression and activity of the mRNA destabilizing protein—tristetraprolin (TTP). But whether corticosteroid-induced MKP-1 acts via p38 MAPK-mediated modulation of TTP function in a pivotal airway cell type, airway smooth muscle (ASM), was unknown. While pretreatment of ASM cells with the corticosteroid dexamethasone (preventative protocol) is known to reduce ASM synthetic function in vitro, the impact of adding dexamethasone after stimulation (therapeutic protocol) had not been explored. Whether dexamethasone modulates TTP in a p38 MAPK-dependent manner in this cell type was also unknown. We address this herein and utilize an in vitro model of asthmatic inflammation where ASM cells were stimulated with the pro-asthmatic cytokine tumor necrosis factor (TNF) and the impact of adding dexamethasone 1 h after stimulation assessed. IL-6 mRNA expression and protein secretion was significantly repressed by dexamethasone acting in a temporally distinct manner to increase MKP-1, deactivate p38 MAPK, and modulate TTP phosphorylation status. In this way, dexamethasone-induced MKP-1 acts via p38 MAPK to switch on the mRNA destabilizing function of TTP to repress pro-inflammatory cytokine secretion from ASM cells. J. Cell. Physiol. 231: 2153–2158, 2016. © 2016 Wiley Periodicals, Inc.

Copyright © 2016 by the American Thoracic Society. Tachyphylaxis of the β2-adrenergic receptor limits the efficacy of bronchodilatory β2-agonists in respiratory disease. Cellular studies in airway smooth muscle (ASM) have shown that inflammatory mediators and infectious stimuli reduce β2-adrenergic responsiveness in a cyclo-oxygenase (COX)-2-mediated, prostaglandin E2 (PGE2)-dependant manner. Herein, we show that sphingosine 1-phosphate (S1P), abioactive sphingolipid thatplays an important role in pathophysiology of asthma, also induces β2-adrenergic receptor desensitization in bronchial ASM cells and exerts hyporesponsiveness to β2-agonists. We treated ASM cells with S1P (1 μM) for up to 24 hours and then examined the temporal kinetics of COX-2 mRNA expression, protein up-regulation, and PGE2 secretion. S1P significantly enhanced COX-2 expression and PGE2 secretion, and this was repressed by the selective COX-2 inhibitor celecoxib, the corticosteroid dexamethasone, or small interfering RNA (siRNA) knockdown of COX-2 expression. In combination with another proinflammatory mediator found elevated in asthmatic airways, the cytokine TNF-α, we observed that S1P-induced COX-2 mRNA expression and protein up-regulation and PGE2 secretion from ASM cells were significantly enhanced. Notably, S1P induced heterologous β2-adrenergic desensitization, as measured by inhibition of cyclic adenosine monophosphate production in response to the short-acting β2-agonist, salbutamol, and the long-acting β2-agonist, formoterol. Taken together, these data indicate that S1P represses β2-adrenergic activity in ASM cells by increasing COX-2-mediated PGE2 production, and suggest that this bioactive sphingolipid found elevated in asthma may contribute to β2-adrenergic desensitization.

A series of dinuclear ruthenium(ii) complexes that contain labile chlorido ligands, [{Ru(tpy)Cl}2{μ-bbn}]2+ {designated Cl-Rubbn; tpy = 2,2′:6′,2′′-terpyridine, bbn = bis[4(4′-methyl-2,2′-bipyridyl)]-1,n-alkane (n = 7, 10, 12, 14 or 16)} and derivatives containing nitro substituents on the tpy ligand and/or secondary amines within the bbn linking chain have been synthesised and their potential as anticancer agents examined. Some of the Cl-Rubbn species showed good anticancer activity against MCF-7 and MDA-MB-231 breast cancer cell lines, with the Cl-Rubb12 complex being four-times more active than cisplatin. Inclusion of nitro substituents on the tpy ligands of Cl-Rubb12 resulted in significantly decreased anticancer activity. The incorporation of amine groups into the linking ligand did not increase the anticancer activity of the Cl-Rubbn complexes. The Cl-Rubbn complexes and those containing amine groups in the linking chain aquated at approximately the same rate, with 50% aquation within 120 minutes. By comparison, the complexes containing nitro substituents on the tpy ligand aquated extremely slowly, with 60% of the chlorido complex remaining 24 hours after they were dissolved in water. Cyclic voltammetry with the model mononuclear complex [Ru{(NO2)3tpy}(Me2bpy)Cl] + {(NO2)3tpy = 4,4′,4′′- trinitro-2,2′:6′,2′′-terpyridine} showed that the nitro substituents exerted a strong effect on the ruthenium centre, with the anodic peak corresponding to the Ru(iii/ii) couple shifted positively by 300 mV compared to that from the non-nitrated parent complex [Ru(tpy)(Me 2bpy)Cl]+. 1H NMR studies of the reaction of the Cl-Rubbn complexes with GMP indicated that the ruthenium complexes covalently bound the nucleotide slowly, with 33% bound in 24 hours. However, the results of this study suggest that the cytotoxicity of the dinuclear ruthenium complexes is a combination of covalent and reversible binding with DNA. © the Partner Organisations 2014.

Background Asthma is a widespread chronic health problem exacerbated by common viral and bacterial infections. Further research is required to understand how infection worsens asthma control in order to advance therapeutic options in the future. Recent research has revealed that β2- Adrenergic receptor (β2-AR) agonists lose bronchodilatory efficacy because the receptor-mediated molecular pathways responsible for their beneficial actions are desensitized by infection. To date, most studies have focussed on viral infection, leaving the impact of bacterial infection on β2-AR desensitization relatively under-investigated. We address this in this study. Methods and Results Utilizing an in vitro model of bacterial exacerbation in airway smooth muscle (ASM) cells, we show that activation of toll-like receptor 2 (TLR2; mimicking bacterial infection) in the presence of an inflammatory stimulus leads to β2-AR desensitization. This occurs via TLR2-dependent upregulation of cyclooxygenase 2 (COX-2) mRNA expression and increased secretion of PGE2. Importantly, PGE 2 causes heterologous β2-AR desensitization and reduces cAMP production in response to short-Acting (salbutamol) and long-Acting (formoterol) β2-Agonists. Thus, bacterial infectious stimuli act in a PGE2-dependent manner to severely curtail the beneficial actions of β2-Agonists. The impact of β2-AR desensitization is demonstrated by reduced gene expression of the critical anti-inflammatory molecule MKP-1 in response to β2-Agonists, as well as impaired bronchodilation in a mouse lung slices. Conclusions Taken together, our results show that, like viruses, bacteria induce prostanoid-dependent β2-AR desensitization on ASM cells. Notably, COX-2 inhibition with the specific inhibitor celecoxib represses PGE2 secretion, presenting a feasible pharmacological option for treatment of infectious exacerbation in asthma in the future. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Mitogen-activated protein kinase phosphatase 1 (MKP-1) represses MAPK-driven signalling and plays an important anti-inflammatory role in asthma and airway remodelling. Although MKP-1 is corticosteroid-responsive and increased by cAMP-mediated signalling, the upregulation of this critical anti-inflammatory protein by long-acting β2-agonists and clinically-used corticosteroids has been incompletely examined to date. To address this, we investigated MKP-1 gene expression and protein upregulation induced by two long-acting β2-agonists (salmeterol and formoterol), alone or in combination with the corticosteroid fluticasone propionate (abbreviated as fluticasone) in primary human airway smooth muscle (ASM) cells in vitro. β2-agonists increased MKP-1 protein in a rapid but transient manner, while fluticasone induced sustained upregulation. Together, long-acting β2-agonists increased fluticasone-induced MKP-1 and modulated ASM synthetic function (measured by interleukin 6 (IL-6) and interleukin 8 (IL-8) secretion). As IL-6 expression (like MKP-1) is cAMP/adenylate cyclase-mediated, the long-acting β2-agonist formoterol increased IL-6 mRNA expression and secretion. Nevertheless, when added in combination with fluticasone, β2-agonists significantly repressed IL-6 secretion induced by tumour necrosis factor α (TNFα). Conversely, as IL-8 is not cAMP-responsive, β2-agonists significantly inhibited TNFα-induced IL-8 in combination with fluticasone, where fluticasone alone was without repressive effect. In summary, long-acting β2-agonists increase fluticasone-induced MKP-1 in ASM cells and repress synthetic function of this immunomodulatory airway cell type. © 2013 Manetsch et al.

Airway remodelling is a consequence of long-term inflammation and MAPKs are key signalling molecules that drive pro-inflammatory pathways. The endogenous MAPK deactivator--MAPK phosphatase 1 (MKP-1)--is a critical negative regulator of the myriad pro-inflammatory pathways activated by MAPKs in the airway.Herein we investigated the molecular mechanisms responsible for the upregulation of MKP-1 in airway smooth muscle (ASM) by the corticosteroid dexamethasone and the β₂-agonist formoterol, added alone and in combination.MKP-1 is a corticosteroid-inducible gene whose expression is enhanced by long-acting β₂-agonists in an additive manner. Formoterol induced MKP-1 expression via the β₂-adrenoceptor and we provide the first direct evidence (utilizing overexpression of PKIα, a highly selective PKA inhibitor) to show that PKA mediates β₂-agonist-induced MKP-1 upregulation. Dexamethasone activated MKP-1 transcription in ASM cells via a cis-acting corticosteroid-responsive region located between -1380 and -1266 bp of the MKP-1 promoter. While the 3'-untranslated region of MKP-1 contains adenylate + uridylate elements responsible for regulation at the post-transcriptional level, actinomycin D chase experiments revealed that there was no increase in MKP-1 mRNA stability in the presence of dexamethasone, formoterol, alone or in combination. Rather, there was an additive effect of the asthma therapeutics on MKP-1 transcription.Taken together, these studies allow us a greater understanding of the molecular basis of MKP-1 regulation by corticosteroids and β₂-agonists and this new knowledge may lead to elucidation of optimized corticosteroid-sparing therapies in the future.

Airway smooth muscle (ASM) is the main regulator of bronchomotor tone. Extensive studies show that in addition to their physical property, human airway smooth muscle (ASM) cells can participate in inflammatory processes modulating the initiation, perpetuation, amplification, and perhaps resolution of airway inflammation. Upon stimulation or interaction with immune cells, ASM cells produce and secrete a variety of inflammatory cytokines and chemokines, cell adhesion molecules, and extracellular matrix (ECM) proteins. These released mediators can, in turn, contribute to the inflammatory state, airway hyperresponsiveness, and airway remodeling present in asthma. As our knowledge of ASM myocyte biology improves, novel bioactive factors are emerging as potentially important regulators of inflammation. This review provides an overview of our understanding of some of these molecules, identifies rising questions, and proposes future studies to better define their role in ASM cell modulation of inflammation and immunity in the lung and respiratory diseases. © 2012.

β 2-agonists are now contraindicated as monotherapy for asthma, and increased PDE4D has been speculated to contribute to this phenomenon. In this study we investigated the expression of PDE4D in asthmatic and nonasthmatic ASM cells and its regulation by formoterol and budes-onide. Primary ASM cells from people with or without asthma were stimulated with transforming growth factor (TGF)- β 1, formoterol, and/or budesonide. PDE4D mRNA was assessed by real-time PCR, or PCR to assess splice variant production. PDE4D protein was assessed by Western blotting, and we investigated the effect of formoterol on cAMP production and PDE activity. Interleukin (IL)-6 was assessed using ELISA. PDE4D mRNA was dose dependently upregulated by formoterol, with a single splice variant, PDE4D5, present. Formoterol did not induce PDE4D protein at time points between 3 to 72 h, whereas it did induce and increase IL-6 secretion. We pretreated cells with actinomycin D and a proteasome inhibitor, MG132, and found no evidence of alterations in mRNA, protein expression, or degradation of PDE4D. Finally PDE activity was not altered by formoterol. This study shows, for the first time, that PDE4D5 is predominantly expressed in human ASM cells from people with and without asthma and that formoterol does not upregulate PDE4D protein production. This leads us to speculate that continual therapy with β 2AR agonists is unlikely to cause PDE4-mediated tachyphylaxis. © 2012 the American Physiological Society.

Airway smooth muscle cells (ASMCs) secrete eotaxin and RANTES (regulated on activation, normal T-cell expressed and secreted) in response to tumour necrosis factor (TNF)-α, which is inhibited by the nuclear factor (NF)-κB inhibitor dimethylfumarate (DMF). NF-κB/IκB (inhibitor of NF-κB) glutathionylation and changes in chromatin remodelling can inhibit NF-κB activity. In this study, we determined whether NF-κB/IκB glutathionylation and reduced histone H3 phosphorylation might underlie the inhibitory effect of DMF on NF-κB activity, and eotaxin and RANTES secretion. Primary human ASMCs were treated with DMF, diamide and/or glutathione (GSH) ethylester (OEt) prior to TNF-α stimulation and were subsequently analysed by ELISA, electrophoretic mobility shift assay, immunofluorescence, co-immunoprecipitation or immunoblotting. DMF reduced intracellular GSH and induced IκBα glutathionylation (IκBα-SSG), which inhibited IκBα degradation, NF-κB p65 nuclear entry and NF-κB/DNA binding. In addition, DMF inhibited the phosphorylation of histone H3, which was possibly mediated by the inhibitory effect of DMF on mitogen- and stress-activated protein kinase (MSK)-1. However, p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase MAPK and MAPK phosphatase-1, upstream of MSK-1, were not inhibited by DMF. Importantly, DMF-mediated effects on NF-κB, histone H3, eotaxin and RANTES were reversed by addition of GSH-OEt. Our data suggest that DMF inhibits NF-κB-dependent eotaxin and RANTES secretion by reduction of GSH with subsequent induction of IκBα-SSG and inhibition of histone H3 phosphorylation. Our findings offer new potential drug targets to reduce airway inflammation in asthma. Copyright©ERS 2011.