Compartmentalized cAMP Signaling in COPD:Focus on Phosphodiesterases and A-Kinase Anchoring Proteins

Cyclic AMP (cAMP) is one of the most effective therapeutic targets in various pulmonary diseases and plays a vital role in relaxing airway smooth muscles and suppressing airway inflammation. However, tolerance in the long-term use and side-effects create the need of more effective approaches. cAMP is able to modulate signaling cascades in a spatio-temporal manner. Therefore, comprehensive understanding of the fluctuations (generation and degradation) of cAMP and its potential functions within certain compartments will most likely help in the screening of novel pharmaceutical targets which have higher efficiency and fewer side effects. In this thesis, Haoxiao Zuo investigated the role of compartmentalized cAMP signaling in a number of structural airway cells of relevance to cigarette smoke exposure using classic pharmacological methods and novel fluorescent tools. Special focus was on phosphodiesterases (PDEs) and A-kinase anchoring proteins (AKAPs). The author found that the expression (mRNA and protein) and activity of both PDE3 and PDE4 were upregulated by cigarette smoke exposure, which likely underpinned alterations of intracellular cAMP levels. In addition, other PDE member, such as PDE8, was a novel target which needed more investigation in the future. In the last part of her thesis, the author showed that AKAPs, with specific focus on Ezrin, Yotiao and AKAP95, played a vital role in maintaining TGF-β1/ cigarette smoke-induced Epithelial-to-mesenchymal transition. Therefore, compartmentalized cAMP signaling being modulated by PDEs and AKAPs represents a promising target to treat airway disorders

Phosphodiesterases as therapeutic targets for respiratory diseases

Chronic respiratory diseases, such as chronic obstructive pulmonary disease (COPD) and asthma, affect millions of people all over the world. Cyclic adenosine monophosphate (cAMP) which is one of the most important second messengers, plays a vital role in relaxing airway smooth muscles and suppressing inflammation. Given its vast role in regulating intracellular responses, cAMP provides an attractive pharmaceutical target in the treatment of chronic respiratory diseases. Phosphodiesterases (PDEs) are enzymes that hydrolyze cyclic nucleotides and help control cyclic nucleotide signals in a compartmentalized manner. Currently, the selective PDE4 inhibitor, roflumilast, is used as an add-on treatment for patients with severe COPD associated with bronchitis and a history of frequent exacerbations. In addition, other novel PDE inhibitors are in different phases of clinical trials. The current review provides an overview of the regulation of various PDEs and the potential application of selective PDE inhibitors in the treatment of COPD and asthma. The possibility to combine various PDE inhibitors as a way to increase their therapeutic effectiveness is also emphasized

Epac Function and cAMP Scaffolds in the Heart and Lung

Evidence collected over the last ten years indicates that Epac and cAMP scaffold proteins play a critical role in integrating and transducing multiple signaling pathways at the basis of cardiac and lung physiopathology. Some of the deleterious effects of Epac, such as cardiomyocyte hypertrophy and arrhythmia, initially described in vitro, have been confirmed in genetically modified mice for Epac1 and Epac2. Similar recent findings have been collected in the lung. The following sections will describe how Epac and cAMP signalosomes in different subcellular compartments may contribute to cardiac and lung diseases

Cigarette smoke exposure alters phosphodiesterases in human structural lung cells

Cigarette smoke (CS), a highly complex mixture containing more than 4,000 compounds, causes aberrant cell responses leading to tissue damage around the airways and alveoli, which underlies various lung diseases. Phosphodiesterases (PDEs) are a family of enzymes that hydrolyze cyclic nucleotides. PDE inhibition induces bronchodilation, reduces the activation and recruitment of inflammatory cells, and the release of various cytokines. Currently, the selective PDE4 inhibitor roflumilast is an approved add-on treatment for patients with severe chronic obstructive pulmonary disease with chronic bronchitis and a history of frequent exacerbations. Additional selective PDE inhibitors are being tested in preclinical and clinical studies. However, the effect of chronic CS exposure on the expression of PDEs is unknown. Using mRNA isolated from nasal and bronchial brushes and lung tissues of never smokers and current smokers, we compared the gene expression of 25 PDE coding genes. Additionally, the expression and distribution of PDE3A and PDE4D in human lung tissues was examined. This study reveals that chronic CS exposure modulates the expression of various PDE members. Thus, CS exposure may change the levels of intracellular cyclic nucleotides and thereby impact the efficiency of PDE-targeted therapies

A-Kinase Anchoring Proteins Diminish TGF-β1/Cigarette Smoke-Induced Epithelial-To-Mesenchymal Transition

Epithelial-to-mesenchymal transition (EMT) plays a role in chronic obstructive pulmonary diseases (COPD). Cyclic adenosine monophosphate (cAMP) can inhibit transforming growth factor-β1 (TGF-β1) mediated EMT. Although compartmentalization via A-kinase anchoring proteins (AKAPs) is central to cAMP signaling, functional studies regarding their therapeutic value in the lung EMT process are lacking. The human bronchial epithelial cell line (BEAS-2B) and primary human airway epithelial (pHAE) cells were exposed to TGF-β1. Epithelial (E-cadherin, ZO-1) and mesenchymal markers (collagen Ӏ, α-SMA, fibronectin) were analyzed (mRNA, protein). ELISA measured TGF-β1 release. TGF-β1-sensitive AKAPs Ezrin, AKAP95 and Yotiao were silenced while using siRNA. Cell migration was analyzed by wound healing assay, xCELLigence, Incucyte. Prior to TGF-β1, dibutyryl-cAMP (dbcAMP), fenoterol, rolipram, cilostamide, and forskolin were used to elevate intracellular cAMP. TGF-β1 induced morphological changes, decreased E-cadherin, but increased collagen Ӏ and cell migration, a process that was reversed by the inhibitor of δ/epsilon casein kinase I, PF-670462. TGF-β1 altered (mRNA, protein) expression of Ezrin, AKAP95, and Yotiao. St-Ht31, the AKAP antagonist, decreased E-cadherin (mRNA, protein), but counteracted TGF-β1-induced collagen Ӏ upregulation. Cigarette smoke (CS) increased TGF-β1 release, activated TGF signaling, augmented cell migration, and reduced E-cadherin expression, a process that was blocked by TGF-β1 neutralizing antibody. The silencing of Ezrin, AKAP95, and Yotiao diminished TGF-β1-induced collagen Ӏ expression, as well as TGF-β1-induced cell migration. Fenoterol, rolipram, and cilostamide, in AKAP silenced cells, pointed to distinct cAMP compartments. We conclude that Ezrin, AKAP95, and Yotiao promote TGF-β1-mediated EMT, linked to a TGF-β1 release by CS. AKAP members might define the ability of fenoterol, rolipram, and cilostamide to modulate the EMT process, and they might represent potential relevant targets in the treatment of COPD

Cigarette Smoke Up-regulates PDE3 and PDE4 to Decrease cAMP in Airway Cells

BACKGROUND AND PURPOSE: 3', 5'-cyclic adenosine monophosphate (cAMP) is a central second messenger that broadly regulates cell function and can underpin pathophysiology. In chronic obstructive pulmonary disease (COPD), a lung disease primarily provoked by cigarette smoke (CS), the induction of cAMP-dependent pathways, via inhibition of hydrolyzing phosphodiesterases (PDEs), is a prime therapeutic strategy. Mechanisms that disrupt cAMP signaling in airway cells, in particular regulation of endogenous PDEs are poorly understood. EXPERIMENTAL APPROACH: We used a novel Förster resonance energy transfer (FRET) based cAMP biosensor in mouse in vivo, ex vivo precision cut lung slices (PCLS), and in human in vitro cell models to track the effects of CS exposure. KEY RESULTS: Under fenoterol stimulated conditions, FRET responses to cilostamide were significantly increased in in vivo, ex vivo PCLS exposed to CS and in human airway smooth muscle cells exposed to CS extract. FRET signals to rolipram were only increased in the in vivo CS model. Under basal conditions, FRET responses to cilostamide and rolipram were significantly increased in in vivo, ex vivo PCLS exposed to CS. Elevated FRET signals to rolipram correlated with a protein upregulation of PDE4 subtypes. In ex vivo PCLS exposed to CS extract, rolipram reversed downregulation of ciliary beating frequency, whereas only cilostamide significantly increased airway relaxation of methacholine pre-contracted airways. CONCLUSION AND IMPLICATIONS: We show that CS upregulates expression and activity of both PDE3 and PDE4, which regulate real-time cAMP dynamics. These mechanisms determine the availability of cAMP and can contribute to CS-induced pulmonary pathophysiology

The novel compound Sul-121 inhibits airway inflammation and hyperresponsiveness in experimental models of chronic obstructive pulmonary disease

COPD is characterized by persistent airflow limitation, neutrophilia and oxidative stress from endogenous and exogenous insults. Current COPD therapy involving anticholinergics, beta(2)-adrenoceptor agonists and/or corticosteroids, do not specifically target oxidative stress, nor do they reduce chronic pulmonary inflammation and disease progression in all patients. Here, we explore the effects of Sul-121, a novel compound with anti-oxidative capacity, on hyperresponsiveness (AHR) and inflammation in experimental models of COPD. Using a guinea pig model of lipopolysaccharide (LPS)-induced neutrophilia, we demonstrated that Sul-121 inhalation dose-dependently prevented LPS-induced airway neutrophilia (up to similar to 60%) and AHR (up to similar to 90%). Non-cartilaginous airways neutrophilia was inversely correlated with blood H2S, and LPS-induced attenuation of blood H2S (similar to 60%) was prevented by Sul-121. Concomitantly, Sul-121 prevented LPS-induced production of the oxidative stress marker, malondialdehyde by similar to 80%. In immortalized human airway smooth muscle (ASM) cells, Sul-121 dose-dependently prevented cigarette smoke extract-induced IL-8 release parallel with inhibition of nuclear translocation of the NF-kappa B subunit, p65 (each similar to 90%). Sul-121 also diminished cellular reactive oxygen species production in ASM cells, and inhibited nuclear translocation of the anti-oxidative response regulator, Nrf2. Our data show that Sul-121 effectively inhibits airway inflammation and AHR in experimental COPD models, prospectively through inhibition of oxidative stress