The Effect of Phosphodiesterase Inhibitors on the Induction of Gene Expression by Long-Acting Beta2-Adrenoceptor Agonists and Glucocorticoids
University of Calgary PRISM: University of Calgary's Digital Repository
Graduate Studies The Vault: Electronic Theses and Dissertations
2013-09-13 The Effect of Phosphodiesterase Inhibitors on the Induction of Gene Expression by Long-Acting beta2-Adrenoceptor Agonists and Glucocorticoids
BinMahfouz, Hawazen
BinMahfouz, H. (2013). The Effect of Phosphodiesterase Inhibitors on the Induction of Gene Expression by Long-Acting beta2-Adrenoceptor Agonists and Glucocorticoids (Unpublished master's thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/28088 http://hdl.handle.net/11023/964 master thesis
University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission. Downloaded from PRISM: https://prism.ucalgary.ca
UNIVERSITY OF CALGARY
The Effect of Phosphodiesterase Inhibitors on the Induction of Gene Expression by Long-Acting beta2-Adrenoceptor Agonists and Glucocorticoids
by
Hawazen BinMahfouz
A THESIS
SUBMITTED TO THE FACULTY OF GRADUATE STUDIES
IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE
DEGREE OF MASTER OF SCIENCE
DEPARTMENT OF MEDICAL SCIENCES
CALGARY, ALBERTA
SEPTEMBER, 2013
© Hawazen BinMahfouz 2013
Abstract
Recently, phosphodiesterases (PDE)4 inhibitors have received approval as a complementary anti-inflammatory treatment for chronic obstructive pulmonary disease
(COPD) patients who are already taking long-acting β2-agonists (LABA)/inhaled corticosteroid (ICS) combination therapy. However, this benefit is seen only in patients of the severe, bronchitic, frequent exacerbator phenotype. Several strategies have been proposed to enhance the clinical efficacy of PDE4 inhibitors. One of these is the use of dual PDE3/4 inhibitors, which in addition to providing superior anti-inflammatory activity when compared to a PDE4 inhibitor alone, will also promote bronchodilatation. The present study demonstrates that a PDE3 plus a PDE4 inhibitor successfully sensitized
BEAS-2B airway epithelial cells transfected with a cyclic adenosine 3',5'- monophosphate (cAMP)-response element luciferase reporter to the LABA, formoterol.
Furthermore, PDE3 plus PDE4 inhibitors in combination prolonged and/or sensitized the ability of formoterol to induce several genes in BEAS-2B cells that have anti- inflammatory potential in the absence and presence of the glucocorticoid, dexamethasone. Collectively, these data suggest that LABA/ICS combination therapy in conjunction with an inhibitors of PDE3 and PDE4 may together improve clinical outcomes in larger a population of severe COPD patients.
ii Acknowledgements
First and foremost, I want to thank God for allowing me to attain my current accomplishments and for enabling so many people to facilitate my Study.
I would also like to express my sincere gratitude to my supervisors, Dr. Robert Newton and Dr. Mark A Giembycz, for their understanding, patience and continual assistance during my Master program. I also appreciate the contributions of my committee members, Dr. Kris Chadee and Dr. Stephen Field, for their kindness and patience throughout my thesis project.
I am grateful for the members of the Newton/Giembycz Lab, Sylvia, Christopher, Elizabeth, Tresa, Suharsh, Taruna and Dong, who provided me with guidance throughout my investigations.
A special thank goes to my father, my King, King Abdullah Bin-Abdulaziz, for his generous scholarship, and the Saudi Cultural Bureau for their support and assistance.
Most importantly, I want to express my sincere appreciation for my parents, whose love has inspired me to accomplish so much. I give many thanks to my sisters, Amal and Nour, as well as to my brothers, Abdullah and Abdulelah, for all of their help with this journey.
I want to recognize the tremendous support and help from my best friend, my sister, Rania Mufti. She has lessened my stress and provided me with friendship, always listening to me. Also, I want to give special thanks to Mahmoud El-Daly for his perpetual assistance.
Finally, I am so grateful for the contributions that each and every one made in giving me both personal and academic assistance.
iii List of Abbreviations
AAT alpha-1 antitrypsin
AC adenylyl cyclase
Ad5 adenovirus serotype 5
AP-1 activator protein-1
ASM airway smooth muscle
ATP adenosine triphosphate
AU adenine–uracil
BSA bovine serum albumin
CaM calmodulin cAMP cyclic adenosine 3',5'-monophosphate
CBP CREB-binding protein
CD cluster of differentiation
CD200 cluster of differentiation 200
CHMP committee for medicinal products for human use
COPD chronic obstructive pulmonary disease
CRE cAMP-response element
CREB cAMP-response element-binding protein
CRISPLD2 cysteine-rich secretory protein LCCL (limulus clotting factor C, cochlin, lgl1) domain-containing 2
CXCL CXC chemokine ligand
CXCR2 CXC chemokine ligand 2 receptor
DAG 1,2-diacylglycerol
DMEM Dulbecco's modified eagle's medium
EC50 half maximal effective concentration
iv EDTA ethylenediaminetetraacetic acid
EMEA European medicines agency
Epac exchange proteins directly activated by cAMP
ERK extracellular signal-regulated protein kinase
FCS foetal calf serum
FEV1 forced expiratory volume in one second
FGF-2 fibroblast growth factor-2
FVC forced vital capacity
GAP GTPase-activating protein
GAPDH glyceraldehyde-3-phosphate dehydrogenase
GDP guanosine diphosphate
GILZ glucocorticoid-inducible leucine zipper
GM-CSF granulocyte/macrophage colony-stimulating factor
GOLD global initiative for chronic obstructive lung disease
GPCR G-protein coupled receptor
GR glucocorticoid receptor
GRE glucocorticoid response element
GRK G-protein receptor kinases
GRO growth-related oncogene
GTP guanosine triphosphate
HAT histone acetyltransferase
HBSS Hanks balanced salt solution
HDAC histone deacetylase
Hsp heat-shock protein
ICAM intercellular adhesion molecules
v ICS inhaled corticosteroid
IFNγ interferon-γ
IL interleukin
IP3 inositol 1,4,5-trisphosphate
IP3R inositol 1,4,5-trisphosphate receptor
JNK c-Jun NH2-terminal kinases
LABA long-acting β2-agonists
LPS lipopolysaccharide
M muscarinic receptors
MAPK mitogen-activated protein kinases
MKP-1 mitogen-activated protein kinase phosphatase-1
MLC myosin light chain
MLCK myosin light chain kinase
MLCP myosin light chain phosphatase
MMP9 matrix metalloproteinase 9
MOI multiplicity of infection
NF-κB nuclear factor κB nGRE negative glucocorticoid response element
NK2 tachykinin neurokinin type 1 p57KIP2 kinase inhibitor protein 2 of 57kDa
PAGE polyacrylamide gel electrophoresis
PDE phosphodiesterases
PEPCK phosphoenol pyruvate carboxykinase
PGE2 prostaglandin E2
PIP2 phosphoinositol 4,5-bisphosphate
vi PKA cyclic AMP-dependent protein kinase A
PKB protein kinase B
PKG cyclic GMP- dependent protein kinase
PKI protein kinase A inhibitor
PLA2 phospholipase A2
PLC phospholipase C
RGS2 regulator of G-protein signalling 2
ROCK Rho-associated protein kinase
SABA short-acting β2-adrenoceptor agonists
Ser serine siRNA small interfering RNA
SLPI secretory leucocyte protease inhibitor
SOCS3 suppressor of cytokine signaling 3
SR sarcoplasmic reticulum
Tc1 type 1 cytotoxic
TGF transforming growth factor
Th1 type 1 helper T cells
TLR4 toll-like receptor 4
TNFα tumor necrosis factor-alpha
TORCH Towards a revolution in COPD health
VEGF vascular endothelial growth factor
WHO World Health Organization
vii Table of Contents
Abstract ...... ii Acknowledgments ...... iii List of Abbreviations ...... iv Table of Contents ...... viii List of Tables ...... xii List of Figures...... xiii
CHAPTER ONE: INTRODUCTION ...... 1 1.1 Chronic obstructive pulmonary disease ...... 1 1.1.1 Definition and prevalence...... 1 1.1.2 Phenotypes of COPD ...... 2 1.1.3 Causes of COPD ...... 2 1.2 Airways inflammation ...... 3 1.3 COPD treatment ...... 4 1.3.1 Bronchodilators in the treatment of COPD ...... 5 1.3.1.1 Anti-cholinergics ...... 5 A. Muscarinic receptors ...... 5 B. Muscarinic receptor signalling ...... 6
1.3.1.2 β2-Adrenoceptor agonists ...... 8
A. β2-Adrenoceptor agonists ...... 8 B. Adrenoceptors ...... 10
C. β2-Adrenoceptor-mediated signalling ...... 11 D. Protein kinase A-independent mechanisms ...... 15
E. β2-Adrenoceptor desensitisation...... 15 1.3.2 Glucocorticoids in COPD ...... 17 1.3.2.1 The glucocorticoid receptor ...... 17 1.3.2.2 Genes activation by glucocorticoids ...... 18 1.3.2.3 Switching off inflammatory gene expression by glucocorticoids ...... 19 1.3.2.4 Post-transcriptional control by glucocorticoids ...... 20
viii 1.3.2.5 Effect of inhaled glucocorticoid in COPD ...... 21 A. Inhaled glucocorticoids as a monotherapy ...... 21
B. Effect of inhaled glucocorticoid with β2-adrenoceptor agonist ...... 22
C. Interaction between glucocorticoid with β2-adrenoceptor agonists ...... 23 1.3.3 Phosphodiesterase 4 (PDE4) inhibitors in the treatment of COPD ...... 26 1.3.3.1 Cyclic nucleotide phosphodiesterases ...... 26 1.3.3.2 Phosphodiesterase 3 ...... 28 1.3.3.3 Phosphodiesterase 4 ...... 28 1.3.3.4 Non-selective phosphodiesterase inhibitors ...... 29 1.3.3.5 Phosphodiesterase 4 inhibitors ...... 29 1.3.3.6 Clinical effects of PDE4 inhibitors ...... 32 1.4 Hypothesis ...... 33 1.4.1 Aims ...... 34
CHAPTER TWO: MATERIALS AND METHODS ...... 35 2.1 Materials ...... 35 2.2 Methods ...... 37 2.2.1 Culture of BEAS-2B cells ...... 37 2.2.2 Luciferase assay ...... 37 2.2.3 Adenoviral over-expression ...... 37 2.2.4 Western blotting ...... 38 2.2.5 RNA extraction, cDNA synthesis and real-time polymerase chain reaction (PCR) ...... 40 2.2.6 Data presentation and statistical analysis ...... 42
CHAPTER THREE: RESULTS ...... 43 3.1 Aim 1: To investigate functional interactions between the LABA, formoterol, and inhibitors of PDE3 and PDE4 on CRE-dependent transcription in BEAS-2B cells...... 43
ix 3.1.1 Rationale ...... 43 3.1.2 Results ...... 43 3.1.2.1 Effect of PDE inhibitors on CRE-dependent transcription ...... 43 3.1.2.2 Effect of PDE inhibitors on formoterol-induced, CRE-dependent transcription ...... 46 3.1.2.3 Effect of PDE3 and PDE4 inhibitors alone and in combination on formoterol-induced, CRE-dependent transcription ...... 49 3.1.2.4 Effect of structurally dissimilar PDE3 and PDE4 inhibitors on BEAS-2B 6xCRE reporter cells ...... 53 3.1.2.5 Role of PKA in PDE inhibitor- and formoterol-induced, CRE- dependent transcription ...... 57 3.1.2.6 Formoterol promotes the phosphorylation of CREB ...... 59 3.1.2.7 PDE inhibitors enhance formoterol-induced, CREB phosphorylation ...... 61 3.2 Aim 2: To investigate functional interactions between formoterol and PDE inhibitors on the induction of putative anti-inflammatory/protective genes in BEAS-2B cells...... 63 3.2.1 Rationale ...... 63 3.2.2 Results ...... 65 3.2.2.1 Effect of formoterol and PDE inhibitors on the expression of anti-inflammatory/protective genes ...... 65 3.2.2.2 PDE inhibitors sensitise BEAS-2B cells to formoterol-induced gene induction ...... 68 3.3 Aim 3: To examine the effect of PDE inhibitors and formoterol on the expression of glucocorticoid-inducible anti-inflammatory/protective genes in BEAS-2B cells...... 71 3.3.1 Rationale ...... 71 3.3.2 Results ...... 71 3.3.2.1 Effect of dexamethasone on the expression of anti- inflammatory/protective genes in combination with cAMP- elevating agents ...... 71
x 3.3.2.2 PDE inhibitors prolong the expression of RGS2 mRNA ...... 76 3.3.2.3 PDE inhibitors sensitise BEAS-2B cells to LABA/glucocorticoid- induced gene induction ...... 78
CHAPTER FOUR: DISCUSSION ...... 81 4.1 Effect of PDE inhibitors on CRE-dependent transcription ...... 82 4.2 Effect of PDE inhibitors on gene expression ...... 83 4.3 The interaction between PDE3 and PDE4 inhibitors ...... 86 4.4 Interaction between PDE inhibitors and LABA ...... 87 4.5 PDE inhibitors and glucocorticoids ...... 89 4.6 Clinical Relevance ...... 94
CHAPTER FIVE: FUTURE WORK...... 98
REFERENCES ...... 100
xi List of Tables
Table 1.1. Classification of PDE enzyme family……………………………………………26 Table 2.1. Western blots antibodies…………………………………………………………39 Table 3.1 Effect of PDE inhibitors on formoterol-induced, CRE-dependent transcription……………………………………………………………………………..……..48 Table 3.2 Effect of inhibition of multiple PDEs on formoterol-induced, CRE-dependent transcription……………………………………………………………………………….……52 Table 3.3 Effect of structurally-dissimilar PDE3 and/or PDE4 inhibitors on CRE- dependent transcription…………………………………………………………………….…56
xii List of Figures
Figure 1.1. Signalling pathway of ASM contraction…………………………………...… 8
Figure 1.2. Classical β2-adrenoceptor signalling pathway in the airways…………… 13 Figure 1.3. Protein kinase A (PKA) induces airways relaxation via different mechanisms ...... 14 Figure 3.1. Effect of PDE inhibitors on luciferase activity in 6xCRE BEAS-2B reporter cells. ……...... ……………………………….. 45 Figure 3.2. Effect of PDE inhibitors and formoterol on luciferase activity in 6xCRE BEAS-2B reporter cells ...... 47 Figure 3.3. Effect of combining inhibitors of PDE3 and PDE4 with formoterol on CRE-dependent transcription ...... 51 Figure 3.4. Effect of structurally-dissimilar PDE inhibitors on CRE-dependent transcription ...... 55 Figure 3.5. Effect of PKIα over-expression on CRE-dependent transcription ...... 58 Figure 3.6. Effect of formoterol on CREB phosphorylation ...... 60 Figure 3.7. Effect of siguazodan and rolipram on formoterol-induced pCREB and pATF-1 ...... 62 Figure 3.8. Effect of LABA and PDE inhibitors on CD200, CRISPLD2, RGS2, MKP-1 and SOCS3 gene expression ...... 67 Figure 3.9. Effect of inhibitors of PDE 3 and PDE 4 on formoterol-inducible genes...... 70 Figure 3.10. Effect of siguazodan, rolipram and formoterol in the presence of dexamethasone on the expression of genes ...... 75 Figure 3.11. Effect of PDE inhibitors on RGS2 expression ...... 77 Figure 3.12. Effect of siguazodan and rolipram on formoterol+dexamethasone- inducible gene expression ...... 80
xiii Chapter One: Introduction
1.1 Chronic obstructive pulmonary disease
1.1.1 Definition and prevalence
Chronic obstructive pulmonary disease (COPD) is a respiratory disorder that is characterized by inflammation of the small airways and lungs. This inflammation leads to irreversible airflow limitation, where the transfer of expired air is decreased in terms of both speed and volume (Pauwels et al., 2001). The accepted diagnosis for COPD is based on the ratio of forced expiratory volume in one second (FEV1) to forced vital capacity (FVC) combined with a relative insensitivity to bronchodilators. If the FEV1/FVC ratio is less than 70%, an individual may be diagnosed as having COPD, especially if that individual is a current or ex-smoker. Based on this ratio, there are four stages of
COPD: patients in the first stage of COPD have an FEV1/FVC ratio of 80%; in the second 50-79%, third stages this ratio fall to 30-49%; and in the fourth, most severe, stage the ratio is less than 30% (Celli and MacNee, 2004; Rabe et al., 2007).
As indicated by these stages, in COPD there is usually a progressive and irreversible decline in lung function produced as a result of chronic exposure to cigarette smoke
(Mannino, 2002) and/or other noxious particulates (Doll et al., 2004; Trupin et al., 2003).
COPD is a common worldwide cause of morbidity and mortality, resulting in significant healthcare costs. Data from the World Health Organization (WHO) demonstrate that
COPD impacts both developed and third-world countries. Specifically, this disease is the fifth leading cause of death in developed countries, representing 3.8% of total deaths, while in underdeveloped countries it is the sixth most common cause of mortality, accounting for 4.9% of all deaths (Lopez et al., 2006). In Canada, more than
1 700,000 individuals have been diagnosed with COPD; however, recent studies suggest that more than 50% of individuals with COPD remain undiagnosed (Vine et al., 2010).
1.1.2 Phenotypes of COPD
There are two broad phenotypes of COPD: a chronic bronchitic phenotype and an emphysematous phenotype that show considerable overlap with each other. Chronic bronchitis involves an inflammation of the bronchial airways. There is enlargement of the mucous glands, increased mucus secretion, defective mucociliary clearance and increased connective tissue deposition, which contributes to increasing the thicknesses of the bronchial walls. Consequently, individuals with bronchitis experience difficulty in breathing because the airflow is obstructed as a result of inflammation and mucus hypersecretion (Jeffery, 1998; Thurlbeck, 1990). This type of COPD is defined by a productive cough that lasts for two periods of at least three months for two (consecutive) years in a row (Stockley, 1995). While, emphysema refers to an unusual enlargement of the air spaces in the lung as a result of a gradual destruction of lung tissue, due to loss of tissue collagen and elastin. In turn, this decreases the pressure driving exhalation and leads to trapped gas in the alveoli (Mattison and Christensen, 2006).
1.1.3 Causes of COPD
There are two main causes of COPD: genetic and environmental. In particular, the only proven genetic cause of COPD is alpha-1 antitrypsin (AAT) deficiency. AAT is an anti- protease that is found in the lungs and protects against the detrimental actions of proteases (Silverman et al., 1998). When the amount of AAT activity is decreased, neutrophil elastase activity is correspondingly increased and this destroys the elastin matrix in the lungs (Stockley, 1995). The main environmental cause of COPD is the
2 continual first-hand or second-hand inhalation of cigarette smoke, which leads to the attraction of inflammatory cells to the lungs that promote inflammation. Other environmental factors that trigger inflammation in COPD include, dust, chemicals and both in- and out-door air pollution (Rabe et al., 2007).
1.2 Airways inflammation
COPD is a chronic inflammatory lung condition that features increased numbers of inflammatory cells, in particular neutrophils and macrophages, in the airways, enhanced expression of cytokines, chemokines, adhesion molecules, inflammatory enzymes and receptors (Barnes, 2008). Several mechanisms may mediate inflammation in COPD.
Thus, cigarette smoke can directly activate inflammatory cells and indirectly induce inflammation via the stimulation of epithelial cells to release chemoattractants and other pro-inflammatory mediators. These increase inflammation by recruiting inflammatory cells to the lung. Acute cigarette smoke exposure leads to the rapid accumulation of neutrophils, which release neutrophil elastase. This mediates elastin degradation and shows the important role of neutrophils in emphysema (Dhami et al., 2000). The increase in neutrophils is associated with an increase in CXC-chemokines, such as
CXC chemokine ligand 1 (CXCL1; aka growth-related oncogene ) and CXCL8 (aka interleukin-8; IL-8). These ligands are potent chemoattractants acting on the CXCR2 receptor expressed by neutrophils to induce neutrophilic inflammation in the airways.
When CXCL8 binds to this receptor, it activates protein kinase B (PKB) and GTPases to promote expression of adhesion molecules (β2-integrins), which facilitate neutrophil migration. In addition, CXCL8 can activate mitogen-activated protein kinases (MAPK) pathways, which may lead to neutrophil degranulation. CXCL1 activation of CXCR2 not
3 only recruits neutrophils, but also attracts inflammatory cells that express CXCR2 such as monocytes, basophils, and T lymphocytes, which are all involved in COPD (Barnes,
2004b).
Cigarette smoke also increases the number of macrophages in the lungs, which differentiate from circulating monocytes and travel to the lungs in response to chemoattractants, such as CCL2 (aka monocyte chemotactic protein-1) acting on
CCR2, and CXCL1 acting on CXCR2 (Traves et al., 2004). Lung macrophages induce
COPD inflammation in part by releasing cytokines and chemokines that attract neutrophils, monocytes and T-cells, and releasing proteases, especially matrix metalloproteinase (MMP) 9 (Barnes, 2004a). Furthermore, although the role of T- lymphocytes in the pathogenesis of COPD is currently unclear, type 1 helper T cells
(Th1) and type 1 cytotoxic T lymphocytes (Tc1) cells are prominent in COPD (Grumelli et al., 2004). These two cell types produce cytokines, such as interferon-γ (IFNγ), which may fulfill a crucial function in inflammation by helping to induce the release of chemokines, such as CXCL9 (aka monokine induced by IFN), CXCL10 (aka IFN induced protein 10) and CXCL11 (aka IFN-inducible T-cell- chemoattractant). These ligands bind to CXCR3 found on Th1 and Tc1 cells and can stimulate the further release of IFNγ. This positive feedback mechanism amplifies inflammatory cell numbers in the lung (Grumelli et al., 2004). Accordingly, enhanced numbers of T-cells releasing IFNγ appear in the lungs of COPD patients (Di Stefano et al., 2004).
1.3 COPD treatment
There is currently no pharmacological therapy that can cure COPD. Rather, current therapies for COPD attempt to manage the symptoms and slow the progression of the
4 disease (Buhl and Farmer, 2005). In general, the pharmacological therapies include bronchodilators, glucocorticoids and, in severe bronchitic patients who suffer frequent acute exacerbations, phosphodiesterase 4 (PDE4) inhibitors.
1.3.1 Bronchodilators in the treatment of COPD
Bronchodilators are the mainstay of COPD management and they exert their effects by relaxing airway smooth muscle (ASM). This alleviates symptoms by improving lung function (as measured by enhanced FEV1 and increased FVC). Some clinical trials have also demonstrated that bronchodilators improve the capacity of an individual to perform exercise and increase their overall health status (Hanania and Donohue, 2007).
Bronchodilators include β2-adrenoceptor agonists (e.g salbutamol, formoterol, salmeterol) and anti-cholinergics (e.g. ipratropium, tiotropium) (Barnes, 2003; Buhl and
Farmer, 2005; Tashkin and Fabbri, 2010).
1.3.1.1 Anti-cholinergics
A. Muscarinic receptors
Anti-cholinergics, or anti-muscarinics, are muscarinic receptor antagonists that inhibit binding of the parasympathetic neurotransmitter, acetylcholine, to muscarinic receptors
(Gross and Skorodin, 1984). There are five subtypes of muscarinic receptors, M1-M5, which are members of the seven transmembrane-spanning, G-protein-coupled receptor
(GPCR), superfamily. M1, M2 and M3 appear to be localized to human airways. These three receptors are mainly found in ASM, proximal airways, and submucosal glands. M1 receptors are also found on parasympathetic ganglia and induce bronchoconstriction when stimulated. The stimulation of M3 receptors promotes bronchoconstriction and mucus secretion because they exist on bronchial smooth muscle and in mucous glands.
5 Alternatively, M2 receptors exist on the ends of cholinergic nerves and when activated inhibit the further release of acetylcholine, and thus function as inhibitory autoreceptors
(Barnes, 2004c; Moulton and Fryer, 2011).
In inflammation, for example in COPD, there is increased release of acetylcholine not only from nerve cells, but also from inflammatory cells, and possibly bronchial epithelial cells (Wessler et al., 1998; Wessler and Kirkpatrick, 2001). Moreover, acetylcholine by enhancing the release of chemokines and cytokines could lead to the attraction of inflammatory cells to the lung (Barnes, 2004c). For example, acetylcholine binds to the
M1 receptor on human bronchial epithelial cells and induces the secretion of monocyte and neutrophil cytokine molecules (Koyama et al., 1992). In addition, secretion of neutrophil chemoattractant molecules, such as leukotriene B4, is induced when the M3 receptor on alveolar macrophages is stimulated by acetylcholine (Profita et al., 2005).
These results demonstrate that acetylcholine may play a role in COPD inflammation and, by blocking M1 and M3 receptors, this inflammation can be reduced. In vitro, studies showed that tiotropium has the ability to inhibit neutrophil migration, which is stimulated by acetylcholine (Buhling et al., 2007). Also, in animal models, inflammation- induced airway remodelling was decreased with tiotropium treatment (Bos et al., 2007).
Taken together, such data imply that tiotropium may have anti-inflammatory effects
(Moulton and Fryer, 2011).
B. Muscarinic receptor signalling
The main muscarinic receptor subtype responsible for bronchial and tracheal smooth muscle contraction is the M3 receptor (Roffel et al., 1990). Acetylcholine binds to M3 receptors and induces a conformational change that promotes association with, and
6 activation of, the heterotrimeric G protein, Gq. This results in the stimulation of the Gqα subunit causing release of guanosine diphosphate (GDP) from Gα and the binding of guanosine triphosphate (GTP) (Billington and Penn, 2003). Furthermore, the active Gα dissociates from Gβγ to subsequently trigger signalling cascades. Activation of Gαq stimulates phospholipase C (PLC) to hydrolyze phosphatidylinositol 4,5-bisphosphate
(PIP2) into 1,2-diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). As a result of
2+ increased IP3 and DAG levels, intracellular Ca is enhanced through its release from internal and extracellular stores. Increasing the level of Ca2+ promotes Ca2+ binding to
2+ calmodulin (CaM) to form a (Ca )4-CaM complex that is able to activate myosin light chain (MLC) kinase (MLCK). MLCK then phosphorylates the 20 kDa MLCs at serine
(Ser)19 causing ASM contraction (Fig. 1.1) (Billington and Penn, 2003). This signalling can be terminated at the level of Gαq by the regulator of G-protein signalling 2 (RGS2), which is a GTPase-activating protein (GAP) that enhances the hydrolysis of GTP to
GDP and, thereby, returns the receptor to its inactive form (Heximer, 2004).
7
Figure 1.1. Signalling pathway of ASM contraction. Interaction of contractile agonists, such as acetylcholine (ACh), with G-protein (Gq)-coupled muscarinic M3 receptors induce phospholipase C (PLC) stimulation. Stimulation of PLC, hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5-trisphosphate (IP3). Subsequently, IP3 binds to IP3 receptor (IP3R) on the sarcoplasmic reticulum (SR), thereby releasing Ca2+ into the cytoplasm. Increasing the level of intracellular Ca2+ 2+ 2+ promotes Ca to bind to calmodulin (CaM). The complex (Ca )4-CaM then activates myosin light chain kinase (MLCK) to phosphorylate the myosin light chain (MLC20), leading to ASM contraction. Adapted from (Giembycz and Newton, 2006).
1.3.1.2 β2-Adrenoceptor agonists
A. β2-Adrenoceptor agonists
β2-Adrenoceptor agonists are administered via the inhaled route (Hanania and
Donohue, 2007). Currently, there are three types of β2-adrenoceptor agonists available for therapy: short, long and ultra-long acting β2-agonists. The short-acting β2- adrenoceptor agonists (SABAs), such as salbutamol and terbutaline, are the initial agents used for the treatment of mild COPD. These drugs possess a rapid onset-of-
8 action, and act to relieve bronchoconstriction within one to three minutes. Consequently, while SABAs represent the ideal choice for the fast relief of bronchoconstriction, their short duration of action, which lasts for a maximum of 6 hours, is a limitation (Sears and
Lotvall, 2005).
The second type of β2-adrenoceptor agonists, the long-acting β2-agonists (LABAs), act for a duration of approximately twelve hours and are given to patients with moderate-to- very severe COPD. Examples of these medications include formoterol and salmeterol; since salmeterol is more lipophilic than formoterol, the latter acts more quickly than the former (Anderson, 1993; Hanania and Donohue, 2007). Specifically, formoterol’s moderately lipophilic status accounts for its ability to readily diffuse across lung tissues
(Anderson, 1993). Most of the inhaled dose that gets to the smooth muscle layer is taken up into the cell membrane, which acts as a depot for the drug. Subsequently, formoterol travels from the cell membrane to interact with the active site of the β2- adrenoceptor. This traveling action may account for the long duration of action of formoterol (Johnson, 2001). Nevertheless, adequate quantities of the drug remain in the aqueous phase outside the cells to facilitate immediate interaction with the β2- adrenoceptor. This interaction cause the rapid onset of action of formoterol with improvements in lung function seen a few minutes after inhalation. Due to its rapid onset, formoterol has received approval as a rescue medication to relieve asthma symptoms (Johnson, 2001).
In contrast to formoterol, salmeterol is considerably more lipophilic (Mason et al., 1991).
Salmeterol is thought to initially enter the cell membrane and then subsequently diffuse within the membrane to access the active site of the β2-adrenoceptor. This process
9 occurs over a period of approximately 30 minutes and accounts for salmeterol’s slower onset of action compared to formoterol (Johnson et al., 1993). Current research asserts that salmeterol’s long duration of action results from its interaction with an anchored binding site, or exo-site, within the β2-adrenoceptor. In particular, salmeterol’s action in binding to this site prevents it from dissociating from the β2-adrenoceptor and helps to create a sustained action (Brittain, 1990). Finally, the new ultra-long acting β2- adrenoceptor agonists, such as indacaterol, induce a quick onset of action and last for
24 hours. The prolonged duration of these new agonists, especially in comparison to other lipophilic β2-adrenoceptor agonists, such as salmeterol, may result from their higher affinity for lipid rafts within the membrane. Furthermore, its relatively rapid onset of action is associated with the high intrinsic efficacy at the receptor level (Baur et al.,
2010; Lombardi et al., 2009).
B. Adrenoceptors
Adrenoceptors belong to the family of GPCRs. These adrenoceptors have been classified into two primary types: α and β. For group α, six subtypes have been identified: α1A, α1B, α1D, α2A/D, α2B and α2C. Upon stimulation, these receptors can cause
2+ contraction by binding to the G-proteins, Gq or Gi, which leads to increased Ca levels and inhibition of adenylyl cyclase (AC) respectively. In contrast, the β family contains only three different receptors, β1, β2 and β3, and the homology between them is ~70%.
The stimulation of these receptors can induce smooth muscle relaxation through Gs activation signalling (Giembycz and Newton, 2006). Furthermore, β3-adrenoceptors can also bind to Gi leading to AC inhibition. However, this effect may not be important in
ASM as there no expression of β3-adrenoceptors, but it may be important for epithelial
10 cell function as those cells possess β3-adrenoceptors (Newnham et al., 1993; Webber and Stock, 1992).
Expression of adrenoceptors in human lung
There are many studies showing that α-adrenoceptors are expressed in the lung, but at the same time, there is no significant evidence for their role in regulating ASM tone
(Anderson, 1995). The expression of β-adrenoceptors has been identified in various cell types within the lung, including airway smooth muscle from trachea to terminal bronchioles (Carstairs et al., 1984). Moreover, β2-adrenoceptors expression is not limited to human ASM, many structural and inflammatory cells have been found to express the β2-adrenoceptors, such as mast cells, macrophages, neutrophils, lymphocytes, eosinophils, epithelial cells, endothelial cells, and both type I and type II alveolar cells (Giembycz and Newton, 2006).
C. β2-Adrenoceptor-mediated signalling
β2-Adrenoceptor agonists initially bind to β2-adrenoceptors associated with a heterotrimeric Gs protein composed of Gsα and Gsβγ subunits, each of which are able to transduce signals to effector molecules. After binding agonist, the receptor undergoes a conformational change. This leads to the disassociation of Gαs from the βγ subunits, allowing Gαs to activate AC (Barisione et al., 2010). Among the nine isoforms of AC, only two isoforms, III and VIII, have not been detected in human ASM cultures (Xu et al., 2001). AC V and VI have been identified as the main forms that fulfill a role in human ASM signalling (Billington et al., 1999; Premont, 1994; Xu et al., 2001). Activated
AC catalyzes the conversion of adenosine triphosphate (ATP) to cyclic adenosine-3', 5'- monophosphate (cAMP). The cAMP then acts as second messenger by interacting with
11 several possible downstream targets of which cAMP-dependent protein kinase (PKA) is the best studied. PKA is a tetrameric enzyme consisting of two catalytic subunits and two regulatory subunits. cAMP binds to the regulatory subunits and alters their conformation allowing the catalytic subunits to be liberated (Billington and Penn, 2003).
The catalytic subunits then phosphorylate a variety of proteins in the cell that induce relaxation of ASM or promote gene expression (Fig.1.2). In ASM, PKA can induce relaxation through several mechanisms: 1) PKA can decrease the release of Ca2+ from the sarcoplasmic reticulum (SR) to the cytosol by phosphorylating the inositol trisphosphate receptor (IP3R) (Supattapone et al., 1988; Tertyshnikova and Fein, 1998).
2) PKA phosphorylates the contractile enzyme MLCK, which lowers the affinity for
19 calcium/calmodulin and subsequently decreases MLC20 phosphorylation at Ser (Conti and Adelstein, 1981; de Lanerolle et al., 1984). 3) PKA inhibits the effect of Rho kinase
(ROCK) by phosphorylating myosin light chain phosphatase (MLCP) at Ser695 and subsequently reducing the ASM tone (Velasco et al., 2002). 4) PKA promotes ASM
1072 hyperpolarization by phosphorylating the BKCa channel at Ser causing an increase in
Ca2+ reuptake and extrusion (Kume et al., 1989) (Fig.1.3). On the other hand, PKA induces gene expression when the catalytic subunits translocate to the nucleus, where they phosphorylate the transcription factor cAMP-response element-binding protein
(CREB) at Ser133 (Gonzalez and Montminy, 1989). Subsequently, activated CREB binds to specific DNA sites, known as cAMP-response elements (CREs), in the promoters of target genes and induces their transcription (Andrisani, 1999). RGS2 is one example of a gene that has been shown to be induced via cAMP/PKA/CREB signalling pathway
12 (Song et al., 2010; Tsingotjidou et al., 2002). This protein may play a significant role in regulating ASM tone (Holden et al., 2011; Xie et al., 2012).
Figure 1.2. Classical β2-adrenoceptor signalling pathway in the airways. β2- Adrenoceptor agonists (A) bind to β2-adrenoceptor (β2-AR) causing activation of adenylyl cyclase (AC), which increases the production of cAMP from adenosine triphosphate (ATP). Subsequently, cAMP binds to the regulatory subunits in protein kinase A (PKA) causing liberation of the catalytic subunits. The catalytic subunits then can induce phosphorylation of 1) different proteins causing airways smooth muscle (ASM) relaxation, or 2) phosphorylation of different transcription factor such as cAMP- response element-binding protein (CREB) which will bind to cAMP-response element (CRE) to induce the expression of genes.
13
Figure 1.3. Protein kinase A (PKA) induces airways relaxation via different 2+ mechanisms 1) PKA can phosphorylate IP3 receptors (IP3Rs) causing inhibition of Ca release from the sarcoplasmic reticulum (SR). 2) Phosphorylation of PKA at myosin light chain kinase (MLCK) induces low binding affinity between Ca2+ and calmodulin (CaM). 3) PKA inhibits the effect of Rho kinase (ROCK) by phosphorylating myosin light chain phosphatase (MLCP) leading to reduce airways smooth muscle (ASM) tone4) The BKCa channel can be phosphorylated by PKA causing ASM hyperpolarization. See text for details.
14 D. Protein kinase A-independent mechanisms
Another target of cAMP is cyclic GMP-dependent protein kinase (PKG). Various studies have shown that cAMP can induce relaxation of smooth muscle cells, including ASM, via an effect on PKG (Hamad et al., 2003; Ward et al., 1995). Moreover, the two exchange-proteins directly activated by cAMP (Epac), are additional cAMP-activated proteins. These are guanine-nucleotide exchange-factors for Ras-like, small GTPases, and have been suggested to result in the activation of MAPK pathway (Bos, 2003; de
Rooij et al., 1998). While studies have demonstrated that Epac binds to Rap and induce signalling through the MAPK pathway (Vossler et al., 1997), this effect of Epac is controversial. Furthermore, this effect probably remains relatively unimportant at pulmonary sites due to the lack of Epac expression in human lungs (Kawasaki et al.,
1998; Vossler et al., 1997). Despite this, more recent data show that human ASM cells in culture express Epac mRNA and the activation of Epac may induce relaxation
(Roscioni et al., 2011).
E. β2-Adrenoceptor desensitisation
Prolonged exposure of ASM cells to β2-adrenoceptor agonists results in receptor desensitisation, which may subsequently cause impaired signalling and limit therapeutic effectiveness (Barisione et al., 2010). Several processes mediate this phenomenon, including phosphorylation and down-regulation of β2-adrenoceptor expression.
Specifically, phosphorylation of the β2-adrenoceptor can be mediated by PKA and a member of another enzyme family collectively referred to as G-protein receptor kinases
(GRK). PKA induces the phosphorylation of Ser and Thr residues in the third intracellular loop of the receptor, causing uncoupling of the receptor. This is considered
15 to be a negative feedback loop. GRK enzymes, in particular GRK2, translocate to the plasma membrane as a consequence of the Gsβγ heterodimers that are released when agonist promotes activation of Gs and induce the subsequent phosphorylation of the β2- adrenoceptor at the C-terminus (Shore and Moore, 2003). This action causes temporary uncoupling of the receptor from Gs. Subsequently, a cofactor called β-arrestin binds to the β2-adrenoceptor and inhibits signalling by preventing further coupling to Gs.
Moreover, β-arrestin acts as adaptor/scaffolding protein to couple the β2-adrenoceptor to the endocytotic machinery components, clathrin and β2-adaptin, causing receptor internalization or sequestration. Two things can happen in the endocytotic machinery; 1) the β2-adrenoceptor can be dephosphorylated, causing recycling of the receptor back to the plasma membrane. 2) The β2-adrenoceptor can be sent to the lysosomes for degradation (Giembycz and Newton, 2006; Shore and Moore, 2003).
Other processes that promote β2-adrenoceptor desensitisation entail downregulation of
β2-adrenoceptor expression via mechanisms such as decreased β2-adrenoceptor transcription (Barisione et al., 2010) and increased post-transcriptional processing of β2- adrenoceptor mRNA (Barisione et al., 2010; Hadcock et al., 1989; Huang et al., 1993).
In addition, prolonged exposure of cells to β2-adrenoceptor agonists may also promote
β2-adrenoceptor desensitisation by up-regulating PDE4 (Seybold et al., 1998). Up- regulation of PDE4 can occur through PKA-dependent activation mechanism. PKA can phosphorylate transcription factors to induce the induction of PDE4 genes or it can phosphorylate pre-existing PDE4 in the cytosol to increase its activity (Giembycz, 1996).
Thus, PDE4 can switch off β2-adrenoceptor signalling by metabolising cAMP to 5’-AMP
(Giembycz, 1996).
16 1.3.2 Glucocorticoids in COPD
Glucocorticoids or corticosteroids are the most effective anti-inflammatory drugs used for several chronic inflammatory diseases, including asthma and rheumatoid arthritis, but are poorly effective in COPD (Barnes, 2010). They act via the glucocorticoid receptor (GR) to promote the expression of anti-inflammatory genes and repress inflammatory gene expression (Barnes, 2010).
1.3.2.1 The glucocorticoid receptor
The GR is a member of the nuclear receptor subfamily 3, which includes receptors for mineralocorticoids, oestrogen, thyroid hormone, retinoic acid and vitamin D (Pujols et al., 2007). The molecular structure of GR is comprised of a variable N-terminal transactivation domain, a DNA binding domain with two zinc finger motifs, and a C- terminal hormone binding domain (Giguere et al., 1986). GR are encoded by a single gene on chromosome 5 in region q31-32, which consists of nine exons. Alternative splicing of the human GR gene generates two main isoforms of GR: GRα and GRβ.
GRα, which consists of 777 amino acids, is the primary isoform that results in glucocorticoid action (Pujols et al., 2007). This isoform is expressed in most tissues and cells. In the lung, the expression of GRα has been identified in the vascular endothelium, airway epithelium, alveolar walls, airway smooth muscle, inflammatory cells and fibroblasts (Adcock et al., 1996; Pujols et al., 2004) as well as in the surface mucosa, submucosal glands, endothelial cells and inflammatory cells of nasal mucosa and nasal polyps (Yun et al., 2002). Alternatively, GRβ, which contains 742 amino acids, cannot bind to glucocorticoids, but attaches to DNA sequences and exerts a dominant negative effect on GRα (Pujols et al., 2007).
17 1.3.2.2 Genes activation by glucocorticoids
Glucocorticoids induce their biological actions by passing through the plasma membrane and binding to specific cytosolic GRs. In their resting state, GRs are usually found in the cytoplasm, where they bind to several heat-shock proteins (Hsp) as well as immunophilins (Stahn et al., 2007). When glucocorticoids bind to the GR complex, a conformational change occurs, resulting in the dissociation of the Hsp and immunophilins and the translocation of free GR from the cytoplasm into the nucleus
(Stahn et al., 2007). Once in the nucleus, the receptor can function as a homodimer, binding to DNA at specific sequences, such as 5’-GGTACAnnnTGTTCT-3’. These sequences are known as glucocorticoid responsive elements (GREs) and are found in the promoter regions of target genes (Starr et al., 1996). Binding of the receptor to GRE sites promotes the recruitment of various co-activators, such as CREB-binding protein
(CBP), which contain intrinsic histone acetyltransferase (HAT) activity that results in the acetylation of core histones (Jenkins et al., 2001; Smirnov, 2002). This acetylation induces chromatin remodelling and the stimulation of RNA polymerase II, leading to gene activation (Deroo and Archer, 2001). Several genes are activated via this mechanism, including genes that have anti-inflammatory effects (Clark, 2007; Clark and
Belvisi, 2012). For example, mitogen-activated protein kinase phosphatase-1 (MKP-1) is a glucocorticoid inducible gene which can play a role in suppressing inflammation through the inhibition of MAPK pathways (Clark, 2003). The MAPK pathways include extracellular signal-regulated protein kinases (ERKs), c-Jun NH2-terminal kinases (JNK) and p38 MAP kinase. Each of these kinases fulfills multiple functions including roles in inflammation, cell proliferation, cell differentiation and, to some extent, ASM contractile
18 responses (Johnson and Lapadat, 2002; Rincón and Davis, 2009). MKP-1 is an important negative regulator of all three of these core MAPK pathways and acts by dephosphorylating the tyrosine and threonine residues that are necessary for MAPK activation (Boutros et al., 2008; Liu et al., 2007). Also, glucocorticoids elevate the expression of lipocortin-1 (Annexin 1), in various cells including pulmonary epithelia, which acts as an inhibitor of phospholipase (PL) A2, causing inhibition of lipid mediator production (Flower and Rothwell, 1994). Moreover, glucocorticoids increase the production of secretory leucocyte protease inhibitor (SLPI) in human airway epithelial cells. SLPI is an antiprotease found in airways and decreases inflammation via its inhibitory effect on inflammatory enzymes such as tryptase (Abbinante-Nissen et al.,
1995)
1.3.2.3 Switching off inflammatory gene expression by glucocorticoids
Glucocorticoids control inflammation by “switching off” the expression of inflammatory genes. Initially, this effect was believed to occur when GR coupled to negative GRE sites (nGRE) and repressed gene transcription (Barnes, 2006a). However, this mechanism failed to explain the anti-inflammatory effect of glucocorticoids due to the fact that simple nGRE sites were not identified in the promoter regions of inflammatory genes (Barnes, 2006a). An alternative mechanism including direct protein–protein interaction has been proposed to explain the anti-inflammatory effect of glucocorticoids.
Activated GRs bind to pro-inflammatory transcription factors, such as nuclear factor κB
(NF-κB) or activator protein-1 (AP-1), which prevent such transcription factors from triggering activation of inflammatory genes transcription (Newton, 2000). The most current mechanisms that explain glucocorticoids’ effects include the interaction of GRs
19 with co-activator molecules, such as CBP. This interaction represses the activity of
HAT, leading to the unwinding of DNA and subsequently inhibiting the expression of inflammatory genes (Barnes, 2006a).
Moreover, switching off inflammation includes the induction of anti-inflammatory genes such as MKP-1 and glucocorticoid-induced leucine zipper (GILZ). These genes have the ability to inhibit an important inflammatory signalling pathway, MAPK, which has the ability to induce inflammatory genes. MKP-1 has the ability to inhibit the three MAPK pathways causing inhibition of many pro-inflammatory genes (Clark, 2003). For example, expression of MKP-1 in ASM induced by dexamethasone inhibited GROα through suppression of JNK signalling pathways (Issa et al., 2007). GILZ, a transcriptional regulator, represses the activities of inflammatory transcription factors including NF-ĸB and AP-1 (Eddleston et al., 2007; Mittelstadt and Ashwell, 2001). For instance, expression of IL-8 was suppressed by increasing GILZ expression in BEAS-
2B cells (Eddleston et al., 2007).
1.3.2.4 Post-transcriptional control by glucocorticoids
Numerous inflammatory genes, such tumor necrosis factor-alpha (TNFα), contain multiple adenine–uracil (AU)-rich sequences in their mRNA, which renders them unstable under normal conditions (Barnes, 2011). However, during inflammation, inflammatory mediators lead to increases in AU-containing mRNA stability (Barnes,
2011). However, glucocorticoids reverse this action of inflammatory mediators, by the induction of mRNA destabilizing proteins, such as, the zinc finger protein, tristetraprolin, which binds to AU-rich areas of mRNAs. Hence, this mediates the decay of mRNAs and
20 the subsequent secretion of inflammatory proteins (Bergmann et al., 2004; Peppel et al.,
1991).
1.3.2.5 Effect of inhaled glucocorticoid in COPD
A. Inhaled glucocorticoids as a monotherapy
Long-term treatment with inhaled corticosteroid (ICS) fails to improve the gradual decrease in FEV1 in COPD patients (Burge et al., 2000; Pauwels et al., 1999). However, it has been reported that daily doses of 40 mg oral prednisone for a period of ten days decreases the recovery time from acute COPD exacerbations (Aaron et al., 2003;
Davies et al., 1999; Niewoehner et al., 1999). The variability among the responses of
COPD patients to ICS may be due to a mixed asthma/COPD phenotype, as subjects with asthma respond more strongly to corticosteroids (Chanez et al., 1997). Despite this, regular treatment with ICS is recommended for COPD patients with a spirometric response to this drug or a FEV1 of under 50% and repeated exacerbations requiring antibiotics treatment. It is recommended that these patients should undergo a 6-week to
3-month trial of ICS to determine the long-term benefits of this medication. However, long-term treatment with oral corticosteroids is not recommended (Pauwels et al., 2001)
In general, corticosteroids are thought to reduce inflammation by stimulating the nuclear enzyme, histone deacetylase (HDAC) 2, to switch off the transcription of inflammatory genes (Barnes, 2006b). This provides an attractive mechanism to explain the poor responsiveness of COPD patients to ICS. Thus, macrophages from COPD subjects have compromised HDAC 2 activity (Ito et al., 2002). Specifically, in vitro research demonstrates that corticosteroids fail to inhibit macrophage production of the neutrophil chemoattractant CXCL8 as well as TNFα and MMP-9, a crucial protease involved in
21 airway modeling (Culpitt et al., 2003). This defect in HDAC activity is thought to be the result of genetics factors, viral infections and/or oxidative stress (Barnes, 2010).
B. Effect of inhaled glucocorticoid with β2-adrenoceptor agonist
Many clinical trials have now demonstrated that therapy combining an ICS with a LABA
(ICS/LABA) is more effective than either treatment alone at reducing exacerbations of
COPD (Nannini et al., 2012). The TORCH (TOwards a Revolution in COPD Health) study investigated combinations of an ICS with a LABA (Vestbo, 2004). In this study, three years of fluticasone propionate/salmeterol xinafoate combination therapy
(fluticasone/salmeterol) decreased moderate to severe exacerbations by 25% in comparison to the placebo, 12% as compared to salmeterol and 9% compared to fluticasone propionate (Calverley et al., 2007). Also, fluticasone/salmeterol resulted in a
31% reduction in exacerbations for individuals with GOLD (Global Initiative for Chronic
Obstructive Lung Disease) stage 2 disease (Jenkins et al., 2009). In a further randomized controlled trial lasting for 44 weeks, COPD patients taking fluticasone/salmeterol experienced 35% fewer exacerbations than those taking salmeterol alone (Kardos et al., 2007). Similarly, a year-long trial showed a 35% decrease in the rate of mild exacerbations for patients taking a budesonide/formoterol combination compared to those taking only budesonide (Szafranski et al., 2003). In a study involving patients with moderate-to-severe COPD, fluticasone/salmeterol exhibited a wider range of anti-inflammatory effects compared with placebo, including significant decrease in biopsy cluster of differentiation (CD8+) cells, (CD45+) leukocytes, and CD4+ cells together with decreases expression of TNFα and IFNγ as well as reduced sputum neutrophils (Barnes et al., 2006). Furthermore, another
22 investigation compared the impact of fluticasone/salmeterol against fluticasone or a placebo (Bourbeau et al., 2007). The preliminary results demonstrated that although each treatment substantially reduced airway inflammation in comparison to the placebo, the anti-inflammatory effect of fluticasone/salmeterol was significantly greater than that for fluticasone. After 12 weeks of treatment with combination therapy, the study showed a decrease in CD8+ T lymphocytes and CD68+ macrophages, both of which may fulfill a significant role in the pathogenesis of COPD (Bourbeau et al., 2007).
C. Interaction between glucocorticoid with β2-adrenoceptor agonists
Glucocorticoids can modulate β2-adrenoceptor functions by reducing the desensitisation of β2-adrenoceptors (Johnson, 2004). Glucocorticoids can stimulate the transcription of
β2-adrenoceptor by binding to the multiple GRE sites found on the promoter area of human β2-adrenoceptor gene and this is consistent with the ability of dexamethasone, a synthetic glucocorticoid, to enhance β2-adrenoceptor mRNA and protein in human lung tissue (Mak et al., 1995). Moreover, glucocorticoids apparently modulate the binding efficiency between the β2-adrenoceptor and Gs; thus, glucocorticoid treatment results in an increase of β2-adrenoceptor–stimulated AC activity and cAMP accumulation (Mak et al., 1995). Pro-inflammatory cytokines, including IL-1β and transforming growth factor
(TGF)-β1 control the binding of β2-adrenoceptor to AC as well as the sensitivity of receptors (Koto et al., 1996). Through their action of decreasing cytokine concentrations, glucocorticoids may prevent or reverse the effects of cytokines
(Johnson, 2004).
While prolonged exposure to β2-adrenoceptor agonists may be pro-inflammatory, this effect can be prevented by glucocorticoids. For instance, β2-adrenoceptor agonists
23 enhance the expression of neurokinin type 1 (NK2)-receptors in airway smooth muscle, potentially increasing bronchoconstrictor responses (Katsunuma et al., 1999). However, glucocorticoids prevent this action via inhibiting the transcription of the NK2-receptor gene (Katsunuma et al., 1998). In addition, there is growing evidence suggesting that
β2-adrenoceptor agonists may independently increase the survival of eosinophils, while glucocorticoids may oppose this effect by increasing apoptosis (Kankaanranta et al.,
2000). Thus, glucocorticoids are capable of preventing many of the adverse inflammatory consequences of chronic β2-adrenoceptor agonist treatment (Barnes,
2002).
In contrast to the effects that glucocorticoids exert on the β2-adrenoceptor and its signalling, which are reasonably well understood, the molecular mechanisms underlying the way in which LABAs affect glucocorticoid action are not. There are data in the literature indicating that LABAs enhance the binding between GR and GRE, which agrees with other evidence showing that in cells overexpressing PKA there, is increased binding of GR to DNA (Rangarajan et al., 1992). Alternatively, LABAs may increase the translocation of glucocorticoid-bound GR from the cytoplasm to the nucleus and that this translocation subsequently causes enhanced glucocorticoid gene transcription
(Eickelberg et al., 1999; Johnson, 2004). Another theory proposes that even in the absence of glucocorticoids, LABAs increase the migration of GR from the cytosol to the nucleus (Eickelberg et al., 1999). Finally, LABAs through cAMP and the activation of
PKA can phosphorylate GR, which may enhance the transcription of some glucocorticoid-inducible genes (Barnes, 2002; Moodley et al., 2013).
24 While the molecular mechanism is not clarified yet, there is good evidence that β2- adrenoceptor agonists may increase the anti-inflammatory effects of glucocorticoids.
For example, the addition of salmeterol increases fluticasone’s inhibition of intercellular adhesion molecules (ICAM)-1 in fibroblasts (Silvestri et al., 2001). Likewise, budesonide’s suppressive effect on granulocyte/macrophage colony-stimulating factor
(GM-CSF) from human airway epithelial cells is enhanced by low concentrations of formoterol (Korn et al., 2001). A 2006 study reported that exposure of BEAS-2B cells to human rhinovirus resulted in increased the expression of two airway modeling factors: fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF)
(Volonaki et al., 2006). While fluticasone and salmeterol both independently repressed this process, the combined effects of these drugs significantly enhanced the inhibitory effects as well as suppressing the chemokine release from BEAS-2B cells triggered by the rhinovirus (Volonaki et al., 2006). Moreover, LABA and ICS combine to synergistically inhibit proliferation by enhancing the expression of the negative cell cycle regulator, p21(Waf1/Cip1) (Roth et al., 2002). Together, these data show that certain anti- inflammatory effects are augmented when a LABA and a glucocorticoid are used in combination when compared to either drug alone. This finding raises the likelihood that because β2-adrenoceptor agonists activate AC to increase cAMP, other cAMP-elevating agents such as PDE inhibitors may act similarly when combined with glucocorticoid (see below).
25 1.3.3 Phosphodiesterase 4 (PDE4) inhibitors in the treatment of COPD
1.3.3.1 Cyclic nucleotide phosphodiesterases
Cyclic nucleotide phosphodiesterases (PDEs) are a large family of enzymes that fulfill a crucial function in cell signalling by hydrolyzing and hence converting the cyclic nucleotides, cAMP and cGMP to 5’AMP and 5’GMP (Bender and Beavo, 2006). In total, there are eleven PDE gene families, PDE1 to PDE11, each of which includes between one and four distinct genes, often with a number of mRNA splice variants that together give rise to more than fifty PDE proteins (Bender and Beavo, 2006; Houslay, 2010).
PDEs are classified according to several factors, including amino acid sequences, structures, enzyme dynamics, regulation modes and tissue distribution. Specifically, the
PDEs that selectively hydrolyse cAMP include PDEs 4, 7 and 8, whereas those that discriminately hydrolyse cGMP include PDEs 5, 6 and 9. The last set of PDEs 1, 2, 3,
10 and 11 hydrolyses both cGMP and cAMP with variable affinity, (Table.1.1 ) (Bender and Beavo, 2006).
Substrates PDE Genes cAMP cGMP Inhibitors
PDE1 A, B and C SCH 57801 PDE2 A BAY 60-7550 PDE3 A and B Siguazodan, Cilostazol PDE4 A, B, C and D Rolipram, Roflumilast PDE5 A Sildenafil PDE6 A, B and C Zaprinast PDE7 A and B BRL 50481 PDE8 A and B PF 4957325 PDE9 A Bay 73-6691 PDE10 A PF 2545920 PDE11 A Non reported
Table 1.1. Classification of PDE enzyme family. Adapted from Bender and Beavo (2006) and Keravis and Lugnier (2011).
26 Nomenclature of PDEs
The nomenclature of PDEs follows several rules. Consider the PDE, HSPDE1A2. The first two letters “HS” prior to the “PDE” indicate the species, in this example, Homo sapiens. The actual term “PDE” signifies a 3,5 cyclic nucleotide phosphodiesterase, and the number following the “PDE,” which ranges from 1-11, denotes its membership to the
PDE1 gene family. Furthermore, the capital letter following the family number signifies the type of gene, which, in the case of the A gene, represents the first member of the family reported in genetic databases. Lastly, the number following the capital letter, the last segment of the name, indicates the variant number reported in the databases
(Bender and Beavo, 2006).
Structure of PDEs
The structure of PDEs can be described as modular, as they contain both catalytic and regulatory domains. PDEs do not share a high sequence identity; however, the catalytic domain is conserved among all 11 families (Xu et al., 2000; Zhang et al., 2004). Thus, the catalytic domain comprises 16 α-helices which are classified into 3 subdomains that create a deep binding pouch for cAMP, cGMP or inhibitors (Zhang et al., 2004). The regulatory domains are located at the N- and C- termini. More specifically, the N- terminus has a membrane targeting domain that assists in regulating cellular and functional compartmentation (Conti and Beavo, 2007). While specific functional roles of the C-terminal region have yet to be determined, one study indicates that this region contributes to PDE dimerization (Kovala et al., 1997).
27 1.3.3.2 Phosphodiesterase 3
Two genes, PDE3A and PDE3B, that show high affinity for cAMP and cGMP, make up the PDE3 family (Reinhardt et al., 1995). Although these genes primarily hydrolyse cAMP, cGMP binds with high affinity and acts as an inhibitor. Therefore, PDE3s are known as cGMP-inhibited cAMP PDEs (Manganiello et al., 1995). PDE3A is located on chromosome 12p12 and yields three splice variants known as PDE3A1, PDE3A2 and
PDE3A3. Conversely, the PDE3B gene is located on chromosome 11p15 and encodes a single product. These two isoforms, which exhibit distinctive tissue distributions, are phosphorylated and activated by PKA and Akt (PKB) (Omori and Kotera, 2007;
Wechsler et al., 2002). While PDE3A appears in the cardiovascular system and the lungs, PDE3B mRNA is expressed in adipose tissue and hepatocytes (Reinhardt et al.,
1995). Small molecules inhibitors of PDE3 have been discovered including cilostamide
(Manganiello et al., 1995; Omori and Kotera, 2007).
1.3.3.3 Phosphodiesterase 4
In the human genome, four genes located on three chromosomes encode the PDE4 isoenzyme family: PDE4A, PDE4B, PDE4C, and PDE4D (Houslay, 2010). The PDE4A and PDE4C genes exist on chromosome 19 at p13.2 and p13.1 respectively, while
PDE4B and PDE4D genes are found on chromosomes 1 at p31 and 5 at q12 respectively (Milatovich et al., 1994; Sullivan et al., 1999; Szpirer et al., 1995). Each gene produces multiple splice variants that can result in the production of more than 20 isoforms (Houslay, 2010). Each PDE4 sub-family have an N-terminal, regulatory domains known as upstream conserved regions, a highly conserved catalytic unit and a
C-terminal region (Lynch et al., 2007).
28 1.3.3.4 Non-selective phosphodiesterase inhibitors
Theophylline
For the past several decades, xanthine compounds, such as theophylline, have been utilized for asthma therapy (Brown, 2007). While the therapeutic impact of theophylline has been ascribed mainly to bronchodilation, lower concentrations of this drug may cause immunomodulatory and anti-inflammatory effects (Sullivan et al., 1994).
Although theophylline has been used extensively to treat respiratory disease, the molecular actions of this drug remain poorly defined. The effectiveness of theophylline treatment in COPD or asthma patients is contributed to non-selective PDE inhibition
(Nicholson and Shahid, 1994). However, when administered at therapeutic doses, theophylline demonstrates negligible non-selective inhibition of PDEs (Boswell-Smith et al., 2006). Moreover, various studies suggest possible mechanisms of action including adenosine receptor antagonism (Yasui et al., 2000) and enhanced HDAC activity (Ito et al., 2002). Regardless, the use of theophylline is limited due to its potentially severe side-effects, which result from its narrow therapeutic index.
Despite the controversy surrounding theophylline, its beneficial clinical effects in airway diseases has encouraged the development of second generation PDE inhibitors in the hope that more effective compounds can be found with reduced side-effects profiles when compared to theophylline (Boswell-Smith et al., 2006).
1.3.3.5 Phosphodiesterase 4 inhibitors
PDE4 represents the main cAMP hydrolyzing enzyme in inflammatory and immune cells, particularly macrophages, eosinophils, and neutrophils, which are all located in
29 the lungs of individuals with COPD and asthma (Brown, 2007). The suppression of
PDE4 activity increases cAMP levels in lung cells to promote anti-inflammatory effects, including the inhibition of cell trafficking and the release of cytokines and chemokines
(Lipworth, 2005). In addition, PDE4 has garnered significant attention due to its expression in ASM (Torphy et al., 1993). Indeed, it was speculated that a selective
PDE4 inhibitor containing both bronchodilator and anti-inflammatory properties should represent a potential therapeutic option for COPD and asthma (Brown, 2007).
Rolipram, a first-generation of PDE4 inhibitor, has been shown to induce anti- inflammatory and anti-immunomodulatory effects in animal models (Vignola, 2004). In spite of its benefits, rolipram has been linked to significant side-effects, such as nausea, vomiting, and gastric acid secretion, caused by the suppression of PDE4 in the central nervous system and parietal glands (Vignola, 2004). To overcome these undesirable effects, several strategies have been proposed to create novel PDE4 inhibitors with an enhanced therapeutic ratio. However, rolipram is still the “gold standard” for studying the biology of PDE4 in experimental systems (Chung, 2006).
Researchers have recently focused their attention on eliminating or modulating the adverse effects of PDE4 inhibitors. Recent studies emphasizing the function of different subtypes suggest that PDE4B may represent the primary subtype mediating anti- inflammatory effects. In contrast, studies in genetically-modified mice indicate that
PDE4D mediates nausea and emesis (Robichaud et al.). In this respect, several studies have shown that cilomilast, a PDE4 inhibitor that progressed to phase III testing in
COPD, is about 10-fold selective for PDE4D and may explain many of the adverse side- effects associated with this compound. Moreover, this explanation may also account for
30 why another PDE4 inhibitor, roflumilast, which fails to differentiate among PDE4 isoenzymes, is better tolerated in humans (Hatzelmann and Schudt, 2001; Manning et al., 1999). However, it is not yet established whether specific targeting of PDE4B subtypes will yield a superior therapeutic ratio for future compounds (Lipworth, 2005).
Despite the efficacy of roflumilast, side-effects of nausea, headache and diarrhoea are still apparent (Fabbri et al., 2009). To resolve this problem several strategies have been proposed. For example, one possibility involves the synthesis of a broader PDE specificity molecule. The future developments of dual specificity compounds that inhibit
PDE4 and other members of the PDE family represent potential benefits for chronic inflammatory lung disease therapy (Banner and Press, 2009; Giembycz and Newton,
2011). Due to the limited scope of this thesis, only PDE3 is discussed in detail.
PDE3 was initially proposed as a possible target for treating asthma and COPD based on the finding that selective PDE3 inhibitors stimulate bronchodilation in humans
(Bardin et al., 1998; Fujimura et al., 1995). As a result, medications that inhibit both
PDE3 and PDE4 could improve lung function by encouraging airway smooth muscle relaxation and blocking inflammation. In addition, PDE3 may also exert desirable effects on the actions of specific pro-inflammatory and immune cells, such as T-lymphocytes, macrophages, monocytes, epithelial cells, endothelial cells, dendritic cells, and airway myocytes, particularly during concurrent PDE4 inhibition, due to the fact that all those cells have been found to co-express PDE3 and PDE4 (Banner and Press, 2009;
Giembycz and Newton, 2011). In vitro studies demonstrate that PDE3 inhibitors exhibit limited repression of T-cell proliferation or IL-2 expression; however, upon the addition of PDE3 inhibitor to cells treated with PDE4 inhibitors, the suppression effects on T-cell
31 proliferation and IL-2 expression were increased remarkably compared to cells treated only with PDE4 inhibitor (Giembycz et al., 1996; Robicsek et al., 1991). Also, the PDE4 inhibitor, rolipram, moderately blocks IL-1β-stimulated GM-CSF release from airway epithelial and A549 cells (Wright et al., 1998). On the other hand, combined therapy involving a dual PDE3/4 inhibitor (ORG-9935) totally inhibited the release of GM-CSF from the airways cells (Wright et al., 1998). Similarly, while the use of PDE3 or PDE4 inhibitors alone failed to inhibit contraction of human airway smooth muscle cells induced by allergen, combination inhibitors worked synergistically to suppress this contraction (Schmidt et al., 2000). Thus, in preclinical models, dual PDE3/4 inhibitors can demonstrate superior efficacy in comparison to compounds that selectively inhibit
PDE4.
Current studies have not yet determined the mechanism(s) responsible for the apparently synergistic effects of dual PDE3/4 suppression (Banner and Press, 2009).
However, reported data demonstrate that PDE3 inhibitors fail to alter the total intracellular cAMP levels in T-lymphocytes (Giembycz et al., 1996). Moreover, these drugs do not result in increased cAMP accumulation in polymorphonuclear cells induced by rolipram. However, investigations have proposed that PDE3 and PDE4 may control distinct pools of cAMP (Denis and Riendeau, 1999).
1.3.3.6 Clinical effects of PDE4 inhibitors
Roflumilast has been recently licensed for use in severe COPD by the European
Medicines Agency (EMEA) Committee for Medicinal Products for Human Use (CHMP)
(Price et al., 2010). Certain benefits have been seen in COPD patients treated with roflumilast. In general, roflumilast treatment enhances lung function and also decreases
32 the rate of mild exacerbations (Profita et al., 2003). Fabbri and colleagues found that moderate to severe COPD patients taking salmeterol, a bronchodilator, experienced improved lung function in response to roflumilast (Fabbri et al., 2009). Additionally, roflumilast also enhanced pre-bronchodilator and post-bronchodilator FEV1, implying that roflumilast’s additive effect on lung function results from mechanisms other than smooth muscle relaxation (Bundschuh et al., 2001; Hatzelmann and Schudt, 2001;
Kumar et al., 2003; Lipworth, 2005; Muise et al., 2002; Spina, 2008). Moreover, it has been reported that not only does PDE4 inhibitors fail to directly affect the smooth muscle of animals but, similar to other selective PDE4 inhibitors, it also lacks significant acute bronchodilator effect in humans (Wollin et al., 2005; Grootendorst et al., 2003).
The enhancement in lung function may be related to its ability to decrease the number of inflammatory cells for COPD patients. Thus, Grootendorst et al., (2007) found that roflumilast treatment diminished the amount and inflammatory activity of neutrophils and eosinophils in induced sputum samples. The anti-inflammatory effect of roflumilast may account for the related improvement in post-bronchodilator FEV1. Therefore, inflammation suppression is likely the mechanism of improvement in lung functioning as induced by roflumilast (Grootendorst et al., 2007).
1.4 Hypothesis
We believe that the anti-inflammatory effect of PDE4 inhibitors and other cAMP- elevating drugs is due, at least in part, to the induction of anti-inflammatory genes. This belief evokes the hypothesis that the enhancement of intracellular cAMP levels in airway structural cells, pro-inflammatory cells and immune cells by inhibitors of
PDE may, in the presence of LABAs, promote, enhance and/or sensitise expression of
33 anti-inflammatory genes. It is further hypothesised that PDE inhibitors can enhance and/or sensitise the expression of glucocorticoid-induced genes.
All studies were conducted using the human bronchial epithelial cell line, BEAS-2B. Due to the fact that it’s a model for human primary airway epithelial cells, induce expression of inflammatory mediators, primary site of action for inhaled anti-inflammatory drugs, and express; β2-adrenoreceptors (Kelsen et al. 1995), PDE3 and PDE4 enzymes (Dent et al. 1998) and GR (Pujols et al., 2001).
1.4.1 Aims
1) To investigate the interaction between the LABA, formoterol, and inhibitors of PDE3 and PDE4 on CRE-dependent transcription in BEAS-2B cells.
2) To investigate the interaction between formoterol and PDE inhibitors, on the induction of putative anti-inflammatory/protective genes in BEAS-2B cells.
3) To examine the effect of PDE inhibitors and formoterol on the expression of glucocorticoid-inducible anti-inflammatory/protective genes in BEAS-2B cells.
34 Chapter Two: Materials and Methods
2.1 Materials
American Type Culture Collection, Rockville, MD, USA: BEAS-2B cells
Bioline Ltd: Taq polymerase, dNTPs
Biotium Inc, Hayward, CA, USA: Luciferase assay kit
Alexis Biochemicals, Enzo Life Sciences, CA-Brockville, Ontario: Bay 60 7550,
BRL 50481
Tocris, Bristol, UK: Siguazodan, rolipram, zardaverine
Dako, Mississauga, ON, Canada: Goat anti-rabbit, goat anti-mouse and rabbit anti- goat immunoglobulins
Fisher Scientific, Nepean, ON, Canada: Methanol, ethanol, sulphuric acid (H2SO4), glacial acetic acid, hydrochloric acid (HCI), sterile tissue culture reagents
Gilead Sciences, Seattle, Washington, USA: GSK 256066
Invitrogen, Burlington, ON, Canada: Dulbecco's Modified Eagle's Medium F-12
(DMEM-F12), fetal calf serum (FCS), 4-12% bis-tris pre-cast gels, Lipofectamine 2000, trypsin ethylenediaminetetraacetic acid (EDTA), penicillin-streptomycin, SYBR GreenER mastermix
Nycomed, Konstanz, Germany: Roflumilast N-oxide
Maybridge, Cronwall, UK: KM03472
Promega, Madison, WI, USA: Reporter lysis buffer, antibiotic G418-sulphate
Thermo Scientific - Pierce Protein Research Products, Rockford, IL, USA: enhanced chemiluminescence (ECL) western blotting substrate
35 Qiagen Ltd, Mississauga, ON, Canada: QlAshredder, RNeasy Mini Kit, DNase set including RDD buffer
Roche Diagnostics, Laval, QC, Canada: Complete protease inhibitor tablets
Sigma-Aldrich Company, Oakville, ON, Canada: TRIZMA-base, L-glutamine, phosphate-buffered saline (PBS), hanks balanced salt solution (HBSS), sodium fluoride
(NaF), sodium pyrophosphate (Na4P2O7), bovine serum albumin (BSA), orange G, dexamethasone, sodium bicarbonate (NaHCO3), polyoxyethylene-sorbitan (Tween20), glycine, Ponceau S, β-mercaptoethanol, acrylamide-bis, TEMED, forskolin, cilostazol, formoterol
36 2.2 Methods
2.2.1 Culture of BEAS-2B cells
Cells were seeded in 12 or 24 well plates in Dulbecco’s modified Eagle’s medium
(DMEM)/Ham’s F12 medium supplemented with 10% fetal calf serum (FCS) and 2mM
L-glutamine and incubated at 37°C in 5% CO2/95% air for 2 or 3 days. At confluence, the cells were growth-arrested for 24h in serum-free medium (SFM) prior to treatments with drugs as indicated.
BEAS-2B cells, previously transfected with a CRE reporter, pADneo2-C6-BGL, containing six tandem CRE motif repeats upstream of a minimal β-globin promoter driving a luciferase gene (6×CRE BEAS-2B cells) (Chivers et al., 2004) were maintained in the presence of 0.4mg/ml G418.
2.2.2 Luciferase assay
After 6h of incubation, cells were harvested in 1x reporter lysis buffer (Promega,
Madison, WI) and subject to one freeze-thaw cycle. Luciferase activity was measured using a Monolight Luminometer (BD Biosciences, San Diego, CA) according to the manufacturer’s instructions.
2.2.3 Adenoviral over-expression
CRE-BEAS-2B cells were grown in 24 well plates until ~ 70% confluence was reached and then were either infected at a multiplicity of infection (MOI) of 30 with Ad5.CMV.Null or Ad5.CMV.PKI expressing adenoviral serotype 5 (Ad5) vectors or left untreated (Meja et al., 2004). The cells were maintained for 24 h at 37°C before replacing the media with serum free media to run the experiments.
37 2.2.4 Western blotting
2.2.4.1 Preparation of cell lysates
Cell lysates in 1 × lamelli buffer (10% glycerol, 5% β-mercaptoethanol, 0.625 M SDS,
0.25 M Tris-HCI pH 6.8 and 0.75 mM bromophenol blue) containing 1 × CompleteTM protease inhibitor mixture and phosphatase inhibitors (50 mM NaF, 2 mM Na3VO4 and
20 mM Na4O7P2) and incubated in -20°C. Cells were scraped from plates and transferred into 1.5 ml Eppendorf tubes, sonicated for 30 min at 4 °C and stored at -20
°C. Prior to polyacrylamide gel electrophoresis analysis, lysates were centrifuged at
2,300 g for 30 sec and boiled for 5 min at 95 ºC.
2.2.4.2 Polyacrylamide gel electrophoresis (PAGE)
The BioRad western blotting system was used for gel electrophoresis. Rainbow protein ladder was loaded (marker for protein size). Proteins were size-fractionated for 1.5 h at
150 mV on 12 % acrylamide gels (0.375 M Tris-HCL pH 8.8, 1 % SDS, 1 % APS, 0.05
% TEMED) in 1 × tris-glycine buffer (0.5 M TRIZMA base, 0.05 % SDS and glycine)
2.2.4.3 Western blotting of gels
Electotransfer of proteins to Hybond-ECL membranes was performed using the BioRad
Mini-cell transfer system at 0.4 mA for 1.5 h in 1x transfer buffer (25 mM TRIZMA base,
191.8 mM glycine, 0.1 % SDS and 20 % methanol). Membranes were stained with
Ponceau S (1.3 mM Ponceau S dissolved in 5 % acetic acid) to display proteins and confirm equal loading.
38 2.2.4.4 Protein detection
After blocking with 5% skimmed milk in 1 × TBS-Tween (0.5 M TRIZMA base, 1.5 M
NaCl, 0.1 % Tween-20) for 1 h at room temperature, membranes were probed with the appropriate primary antibody. Primary antibodies were either incubated for 1 h at room temperature in TBS-Tween or overnight at 4°C in TBS-Tween plus 5% BSA with gentle shaking. After 3 washes, membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody in TBS-Tween (1 h at room temperature).
Again membranes were washed 3 times before detection using ECL. Autoradiography was used to detect the presence of bands. In all experiments, TBS/Tween was used for membranes washing.
2.2.4.5 Stripping and reprobing
Membranes were submerged in stripping buffer (0.5 M Tris-HCl, pH=6.8, containing
0.1M β-mercaptoethanol and 10% SDS) and incubated at 70 ºC for 5 min with shaking.
After washing 3 x 5 min membranes were subjected to immunodetection as described above.
PrimaryAntibody Secondary Dilution Company Cat. number P-CREB (Ser133) Rabbit 1:1000 Cell Signaling Technology 9191
CREB Rabbit 1:1000 Cell Signaling Technology 9197
GAPDH Mouse 1:40000 AbD Serotec 4699-9555
Table 2.1. Western blots antibodies
39 2.2.5 RNA extraction, cDNA synthesis and real-time polymerase chain reaction
(PCR)
2.2.5.1 RNA extraction
Total RNA was extracted from cells using the RNeasy mini kits according to the manufacturer’s guidelines. The Medium was removed and cells were lysed with RLT buffer containing 1% β-mercaptoethanol (plates were stored in -80 °C). Cells were subject to 1 freeze-thaw cycle before scraping from plates and the lysate homogenized using a QlAshredder. The homogenized lysate was mixed with an equal volume of 70% ethanol and transfered to RNeasy spin column. Following centrifugation for 30 s at 8000 g, the column was washed with RW1 buffer (350 µl) and respun at 30 s at 8000 x g. To remove contaminating genomic DNA, the column was incubated with 80 μl of 1 × RDD buffer containing 18 units of DNase I for 30 min at room temperature. After the 30 min incubation, the column was washed again with RW1 (350 μl) and centrifuged for 30 s at
8000 x g. RPE buffer was added (500 µl) to the spin column and centrifuged for 30 s at
8000 x g (this step was repeated once). Finally, RNA was eluted by adding 35 μl of
RNase free water and centrifuged for 1 min at 8000 x g. To assess the purity and the concentration of RNA samples, the ratio of the absorbance at 260 and 280nm was used.
2.2.5.2 cDNA synthesis
Total RNA, 0.5 µg, was reverse-transcribed using avian myeloblastosis virus (AMV) reverse transcriptase (1 U), 1 mM dNTPs, 1 U RNase inhibitor and 5 ng random primers were added to 0.2 ml thin walled tubes. Thermocycler instrument was adjusted with the
40 following parameters 70°C for 5 min, 37°C for 1 h and 94°C for 4 min to generate the cDNA. The product was later diluted (1:5) in RNase-free water and stored at 4°C.
2.2.5.3 Real-time PCR
Real-time PCR was performed using StepOne instrument. 2.5 µl of each cDNA samples were added to MicroAmp optical 96 well reaction plates. Followed by adding 5 μl SYBR green ER MasterMix, 2.3 μl water and 2.5 ng of reverse and forward primers. All samples were analyzed in duplicate. Later plates were spun and mixed before putting them in the machine with the following condition: 50 °C for 2 min, 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. In order to assess primer specificity, a dissociation curve was produced after amplification was complete (95 °C for 15 s, 60 °C for 20 s, and 95 °C for 15 s with ramping to 95 °C over 20 min). Relative standard curves for target genes were constructed from a serial dilution of cDNA containing the target gene. Results were normalized to the housekeeping gene GAPDH.
Primers were as follows:
GAPDH Forward 5′-TTC ACC ACC ATG GAG AAG GC-3′
Reverse 5′-AGG AGG CAT TGC TGA TGA TCT-3′
RGS2 Forward 5′-CCT CAA AAG CAA GGA AAA TAT ATA CTG A-3′
Reverse 5′-AGT TGT AAA GCA GCC ACT TGT AGC T-3′
MKP-1 Forward 5′-GCT CAG CCT TCC CCT GAG TA-3′
Reverse 5′-GAT ACG CAC TGC CCA GGT ACA-3′
CRISPLD2 Forward 5’-CAA ACC TTC CAG CTC ATT CAT G-3’
41 Reverse 5’-GGT CGT GTA GCA GTC CAA ATC C-3’
CD200 Forward 5’-GGA CTG TGA CCG ACT TTA AGC AA-3’
Reverse 5’-AGC AAT AGC GGA ACT GAA AAC C-3’
DNA synthesis lab at the University of Calgary, synthesized the RT-PCR primers.
2.2.6 Data presentation and statistical analysis
Data are presented as means ± S.E of ‘n’ independent determinations. Total LabTM software (Nonlinear Dynamics, Newcastle, UK) was used to quantify the density of each band. Comparison between groups of experimental was analyzed statistically using
Student's paired t test or one-way analysis of variance (ANOVA) with a Bonferroni or
Dunnet’s post-test (GraphPad Prism, version 5.03). Significance between groups was assumed when P < 0.05 (*), P < 0.01 (**) or P < 0.001 (***).
42 Chapter Three: Results
3.1 Aim 1: To investigate functional interactions between the LABA, formoterol, and inhibitors of PDE3 and PDE4 on CRE-dependent transcription in BEAS-2B cells.
3.1.1 Rationale
Current research provides evidence that the clinical actions of glucocorticoids are due, in part, to the transactivation by agonist-bound GR of several anti-inflammatory genes
(Newton and Holden, 2007). In a similar way, it is possible that PDE inhibitors alone or in combination with a LABA could also exert beneficial effects in COPD by a transcriptional activation process through the activation of CREB or other cAMP- activated related transcription factors (Xie et al., 2011). Given that PDE inhibitors and
LABAs work by increasing cAMP, additivity or even synergy may be produced when these drugs are used in combination. Thus, in this aim of the project, the possible interaction between the LABA, formoterol, and selective inhibitors of PDE3 (e.g. siguazodan), PDE4 (e.g. rolipram), and other PDE inhibitors including the PDE2 inhibitor, Bay 60-7550, and the PDE7 inhibitor, BRL 50481, will be investigated. In these studies, BEAS-2B cells, stably transfected with a validated cAMP response element (CRE)-dependent luciferase reporter (Himmler et al., 1993; Meja et al., 2004), are used as a surrogate of cAMP-induced, anti-inflammatory gene expression.
3.1.2 Results
3.1.2.1 Effect of PDE inhibitors on CRE-dependent transcription
The effect of different PDE inhibitors was investigated on CRE-dependent transcription.
BEAS-2B 6CRE reporter cells were cultured with various concentrations of the PDE2
43 inhibitor, Bay 60-7550, the PDE3 inhibitor, siguazodan, the PDE4 inhibitor, rolipram, the general PDE7 inhibitor, BRL 50481 and the PDE7B selective inhibitor, KM 03472. A fixed concentration (10 µM) of the AC activator, forskolin, was also incorporated into the study protocol as a positive control. After 6 h, the cells were harvested for luciferase assay. In each case, the PDE inhibitors were very weak activators of CRE-dependent transcription, whereas forskolin produced a 12-20-fold induction reporter activity (Fig.
3.1). These data can be interpreted in three possible ways. First, the cAMP produced by
PDE inhibitors does not regulate CRE-dependent transcription. Second, the basal synthesis of cAMP is very low, such that inhibiting cAMP metabolism with the PDE inhibitors does not increase cAMP to a level necessary to achieve significant CRE- dependent transcription. Third, cAMP-induced, CRE-dependent transcription is regulated by more than one PDE, such that inhibition of a single PDE enzyme only has a relative modest effect on luciferase expression.
44
A B C 40 40 40 N= 3 N= 3 N= 5 20 20 20
3 3 3
2 2 2 (foldinduction)
1 1 1 CRE Reporter CRE Activity
0 0 0 -9 -8 -7 -6 -5 -4 FSK -9 -8 -7 -6 -5 -4 FSK -9 -8 -7 -6 -5 -4 FSK Log [Bay 60-7550 (M)] Log [Siguazodan (M)] Log [Rolipram (M)]
D E 40 40 N= 4 N= 3 20 20
3 5 4 2 3
(foldinduction) 2 1 CRE Reporter CRE Activity 1 0 0 -9 -8 -7 -6 -5 -4 FSK -9 -8 -7 -6 -5 -4 FSK Log [BRL 50481 (M)] Log [KM 03472 (M)]
Figure 3.1. Effect of PDE inhibitors on luciferase activity in 6xCRE BEAS-2B reporter cells. Cells were treated with various concentrations (1 nM to 30 µM) of A) Bay 60-7550, B) Siguazodan, C) Rolipram, D) BRL 50481 or E) KM 03472. A fixed concentration of forskolin (FSK;10 µM) was used as appositive control. Cells were harvested after 6 h for luciferase assay. Data (n = 3 – 5 as indicated in each panel), expressed as fold induction of luciferase relative to unstimulated cells, are plotted as means ± SE.
45 3.1.2.2 Effect of PDE inhibitors on formoterol-induced, CRE-dependent transcription
To examine possible interactions between PDE inhibitors and the LABA, formoterol, on
CRE-dependent transcription, BEAS-2B CRE reporter cells were pre-treated for 30 min with the highest concentration of each PDE inhibitor that retains selectivity for its target enzyme (Moodley et al., 2013). The PDE2 inhibitor, Bay 60-7550 (1 µM), PDE3 inhibitor, siguazodan (10 μM), PDE4 inhibitor, rolipram (10 μM), PDE7 inhibitor, BRL
50481 (30 µM), or PDE7B inhibitor, KM 03472 (10 µM), were applied to the cells and then concentration-response curves to formoterol (1 pM to 10 nM) were constructed. As shown in figure 3.2, formoterol alone activated CRE-dependent transcription in a concentration-dependent manner with an EC50 value of approximately 100 pM (Table
3.1). In the presence of siguazodan or rolipram, the formoterol concentration-response curves were significantly, but modestly, displaced to the left (approximately 5-fold) without any change in the maximum response (Table 3.1). KM 03472 also produced a slight and significant leftwards displacement of formoterol concentration-response curve
(approximately 1.2-fold) and, in addition, enhanced the maximum formoterol-induced response (from 19- to 23-fold; Table 3.1). Similarly, BRL 50481 augmented the maximal formoterol-induced response (from 19- to 27-fold) without affecting its potency (Table
3.1). In contrast, Bay 60-7550 displaced formoterol concentration-response curves to the right (by 1.6-fold) (Fig. 3.2 A; Table 3.1).
46 A B 15 Naive Naive + BAY 60-7550 + Siguazodan 10 10
5
5
(fold induction) CRE ReporterCRE Activity
N = 4 N = 7 0 0
-12 -11 -10 -9 -8 -12 -11 -10 -9 -8
NS NS
Log [Formoterol (M)] Sig Log [Formoterol (M)] Bay C D 20 Naive 30 Naive + Rolipram + BRL 50481 15
20 10
(fold induction) 5 10 CRE ReporterCRE Activity
N = 8 N = 6 0 0
-12 -11 -10 -9 -8 -12 -11 -10 -9 -8
NS NS
Roli Log [Formoterol (M)] Log [Formoterol (M)] BRL
E 30 Naive + KM03472
20
10
(fold induction) CRE ReporterCRE Activity
N = 6 0
-12 -11 -10 -9 -8 NS
Log [Formoterol (M)] KM
Figure 3.2. Effect of PDE inhibitors and formoterol on luciferase activity in 6xCRE BEAS-2B reporter cells. BEAS-2B 6CRE reporter cells were pre-incubated for 30 min with vehicle or the PDE inhibitors: A, Bay 60-7550 (1 μM); B, siguazodan (10 μM); C, rolipram (10 μM); D, BRL 50481 (30 μM); or E, KM 03472 (10 μM). Formoterol at various concentrations was then added and after 6 h the cells were harvested for luciferase measurement. Data (n= 4 for A; n= 7 for B; n= 8 for C; n= 6 for D; n= 6 for E), expressed as fold induction, are plotted as mean ±S.E.
47 Treatment
logEC50 Fold Fold logEC50 β2-adrenoceptor (M) induction paired t- PDE inhibitors paired t-test agonist (± SE) (± SE) test N
-10.1 7.7 - ±0.06 ±0.3 Formoterol P < 0.04 4 -9.8 7.5 NS +Bay 60-7550 * ±0.07 0.2 -10.05 11.5 - 0.03 ±1.2 Formoterol 7 P < 0.0001 -10.33 12.1 NS +Siguazodan *** ±0.04 ±1.4 -9.9 16.9 - ±0.02 ±1.4 NS Formoterol 8 P < 0.0001 -10.23 17 +Rolipram *** ±0.05 ±1.5 -9.9 19.1 - ±0.02 ±1.1 P < 0.0002 Formoterol 6 NS -10.01 27 *** +BRL 50481 ±0.02 ±1.8 -9.9 19.4 - ±0.03 P < 0.005 ±2.4 P < 0.0006 Formoterol 6 -10.1 ** 23.3 *** +KM 03472 ±0.05 ±2.2
Table 3.1. Effect of PDE inhibitors on formoterol-induced, CRE-dependent transcription. CRE BEAS-2B cells were treated with various concentration of formoterol in the absence or the presence of Bay 60-7550 (1 µM), siguazodan (10 µM), rolipram (10 µM), BRL 50481 (30 µM) or KM 03472 (10 µM). Cells were harvested after
6 of incubation for luciferase measurement. Data originate from figure 3.2. LogEC50 values and the fold induction were analysed by Student’s paired t-test.
48 3.1.2.3 Effect of PDE3 and PDE4 inhibitors alone and in combination on formoterol-induced, CRE-dependent transcription
The preceding data, with the exception of PDE2 inhibitor, Bay 60-7550, demonstrated the presence of functional interactions between each PDE inhibitor and LABA. Although the functional effect (the shifts in logEC50) was relatively small, they were, nevertheless, statistically significant. This suggests that the pool of cAMP that is elevated by formoterol, and which leads to activation of the CRE reporter, is also subject to degradation by these PDEs. However, as the ability of each PDE inhibitor alone to modulate the response to formoterol was small, we sought to explore the possibility that inhibition of multiple PDEs could elicit a greater effect than the inhibition of a specific
PDE family alone. For the purpose of this study, we decided to continue with inhibitors of PDE3 and PDE4 because they represent the main cAMP hydrolysing activities in
BEAS-2B cells and in primary airway epithelial cells (Dent et al., 1998; Fuhrmann et al.,
1999). CRE BEAS-2B cells were pre-treated for 30 min with maximally effective concentrations of PDE3 inhibitor, siguazodan (10 μM) and/or PDE4 inhibitor, rolipram
(10 μM), and then concentration-response curves to formoterol (1 pM to 10 nM) were constructed. In the absence of formoterol, siguazodan alone did not significantly activate CRE-dependent transcription, while rolipram produced a 1.6-fold induction of the reporter (Fig. 3.3 A). However, when siguazodan and rolipram were used in combination, there was a significant (approximately 3-fold) induction of the CRE reporter (Fig. 3.3 A). Formoterol concentration-response curves with fixed concentration of siguazodan or fixed concentration of rolipram were displeased to the left upon the addition of rolipram or siguazodan by approximately 4-fold (Fig. 3.3 B and C; Table 3.2).
49 Moreover, the formoterol concentration-response curve was significantly displaced to the left by approximately 10-fold upon the addition of siguazodan and rolipram in combination, which was greater than that produced by either siguazodan or rolipram alone (Fig. 3.3 D; Table 3.2).
50 A
4 N = 10 *** *** 3 ***
2 ns **
(fold induction) 1 CRE ReporterCRE Activity
0 _ _ Siguazodan + + Rolipram _ _ + +
B C D
12 Naive Naive 30 Naive + Roli 12 + Sig + Sig+Roli
9 9 20
6 6
10 (fold induction)
3 3 CRE ReporterCRE Activity
N = 6 N = 4 N = 6 0 0 0
-12 -11 -10 -9 -8 -12 -11 -10 -9 -8 -12 -11 -10 -9 -8
NS
Sig Roli
Log [Formoterol (M)] Log [Formoterol (M)] Log [Formoterol (M)]
Sig+Roli Roli+Sig + Siguazodan Roli+Sig + Rolipram
Figure 3.3. Effect of combining inhibitors of PDE 3 and PDE4 with formoterol on CRE-dependent transcription. CRE BEAS-2B reporter cells were not simulated or stimulated for 30 min with siguazodan (10 μM) (Sig), rolipram (10 μM) (Roli), or a combination of both together (both 10 μM) (Sig + Roli) (A). Data (n = 6) expressed as fold induction, relative to unstimulated cells, are plotted as mean ±S.E. Statistical significance, relative to unstimulated, using ANOVA with a Bonferroni post-test is indicated; ** P < 0.01 and *** P < 0.001 for (A). Alternatively, cells were pre-treated for 30 min with siguazodan (10 μM) (Sig), rolipram (10 μM) (Roli), or a combination of both together (both 10 μM) (Sig + Roli) before treating with various concentrations of formoterol, after 6h, cells were harvested for luciferase measurement. Data (n= 6 for B; n= 4 for C; n= 6 for D) expressed as fold induction, are plotted as mean ±S.E.
51 Treatment
logEC Fold Fold 50 logEC β -adrenoceptor 50 2 (M) induction paired t- PDE inhibitors paired t- agonist (± SE) (± SE) test N test
-10.54 8.2 +Siguazodan ±0.04 ±0.8 Formoterol 5 P < 0.0001 +Siguazodan -11.05 10.2 NS +Rolipram ±0.05 *** ±0.6 -10.87 11.04 +Rolipram ±0.08 ±0.98 NS Formoterol 4 P < 0.04 +Siguazodan -11.20 11.7 +Rolipram ±0.1 * ±1.3 -9.97 21.6 - ±0.02 P < 0.0001 ±0.99 Formoterol 6 NS + Siguazodan -10.75 *** 23.3 +Rolipram ±0.09 ±1.3
Table 3.2. Effect of inhibition of multiple PDEs on formoterol-induced, CRE- dependent transcription. As illustrated in figure 3.3, cells were pre-incubated with siguazodan, rolipram or both. After 30 min cells were treated with various concentration of formoterol and 6 h later, cells were harvested for luciferase activity. LogEC50 values and the fold activation were analysed by Student’s paired t-test.
52 3.1.2.4 Effect of structurally dissimilar PDE3 and PDE4 inhibitors on BEAS-2B
6xCRE reporter cells
To validate our findings that inhibition of PDE3 and/or PDE4 sensitise LABA-induced
CRE-dependent transcription, we conducted additional experiments with structurally dissimilar PDE3 and PDE4 inhibitors on luciferase activity in 6xCRE BEAS-2B reporter cells. Cells were incubated with various concentrations of the PDE3 inhibitor, cilostazol, the PDE4 inhibitors, GSK 256066 or roflumilast N-oxide, or the dual PDE3/4 inhibitor, zardaverine. As before, the AC activator, forskolin (10 μM), was used as a positive control and the cells were harvested after 6 h for luciferase assay. As was the case with the preceding data, inhibitors of PDE3 and PDE4 demonstrated extremely weak, or no, activation of CRE-dependent transcription (Fig. 3.4 A-C). By comparison the dual
PDE3/4 inhibitor, zardaverine, induced almost a 3-fold induction of reporter activity (Fig.
3.4 D) and forskolin produced a 12- to 20-fold induction of the CRE-dependent transcription (Fig 3.4 A-D).
Furthermore, CRE BEAS-2B cells were pre-incubated for 30 min with maximally- effective concentrations of the PDE3 inhibitor, cilostazol (10 µM), PDE4 inhibitors, GSK
256066 (10 nM) or roflumilast N-oxide (10 nM) or the dual PDE3/4 inhibitor, zardaverine
(10 µM). Subsequently, concentration-response curves to formoterol (1 pM to 10 nM) were constructed. The results demonstrated that formoterol activated CRE-dependent transcription in a concentration-dependent manner with an EC50 of approximately 40 pM
(Fig 3.4 E-H; Table 3.3). In the presence of cilostazol, GSK 256066 or roflumilast N- oxide, the formoterol concentration-response curves were displaced (approximately 2- fold) to the left without any change in the maximum response (Fig 3.4 E-G; Table 3.3).
53 By contrast, the dual-selective inhibitor, zardaverine, significantly displaced the formoterol concentration-response curves to the left by approximately 6.3-fold (Fig. 3.4
H; Table 3.3).
54
A B C D 40 40 40 40 N= 6 N= 4 N= 6 N= 6 20 20 20 20
3 3 3 3
2 2 2 2
1 1 1 1
CRE ReporterCRE Activity (fold induction)
0 0 0 0 -9 -8 -7 -6 -5 -4 FSK -11 -10 -9 -8 -7 -6 -5 FSK -10 -9 -8 -7 -6 -5 FSK -9 -8 -7 -6 -5 -4 FSK Log [Cilostazol (M)] Log [GSK 256066 (M)] Log [Roflumilast N-oxide (M)] Log [Zardaverine (M)]
E F G H 25 30 30 Naive Naive Naive Naive 20 + Cilostazol + GSK256066 + Roflumilast N-oxide + Zardaverine 20 20 20 15
10 10
10 10 (fold induction)
5 CRE ReporterCRE Activity
N = 6 N = 6 N = 6 N = 5 0 0 0 0
-12 -11 -10 -9 -8 -12 -11 -10 -9 -8 -O -12 -11 -10 -9 -8 -12 -11 -10 -9 -8
NS
NS
NS
NS N
Log [Formoterol (M)] Log [Formoterol (M)] Log [Formoterol (M)] Zar Log [Formoterol (M)]
GSK
Rof Cilo
Figure 3.4. Effect of structurally-dissimilar PDE inhibitors on CRE-dependent transcription. BEAS-2B 6×CRE reporter cells were pre-incubated for 30 min with various concentrations of A) cilostazol, B) GSK 256066, C) roflumilast N-oxide or D) zardaverine, alone, and FSK (10 µM). Alternatively, cells were pre-stimulated for 30 min with vehicle, cilostazol (E; 10 μM) (Cilo), GSK 256066 (F; 10 nM) (GSK), roflumilast N- oxide (G; 10 nM) (Rof N-O), or zardaverine (H; 10 μM) (Zar) before adding various concentrations of formoterol. After 6 h, cells were harvested for luciferase measurement. Data (n = 4 - 6), expressed as fold induction, are plotted as mean ±S.E.
55
Treatment logEC (M) logEC Fold Fold β - 50 50 2 PDE N (± SE) paired t-test induction paired t- adrenoceptor inhibitors (± SE) test agonist -10.5 16 - ±0.1 P < 0.04 ±0.8 Formoterol 6 NS -10.8 * 18.5 +Cilostazol ±0.2 ±1.4 -10.3 18.47 - ±0.06 P < 0.0007 ±0.5 Formoterol 6 NS +GSK -10.7 *** 18.9 256066 ±0.09 ±0.4 -10.3 17.6 - ±0.06 P < 0.014 ±0.97 Formoterol 6 NS +Roflumilast -10.6 * 21 N-oxide ±0.1 ±2.18 -10.5 15.7 - ±0.14 P < 0.0001 ±1.7 Formoterol 5 NS -11.2 *** 19.3 +Zardaverine ±0.11 ±2.6
Table 3.3. Effect of structurally-dissimilar PDE3 and/or PDE4 inhibitors on CRE- dependent transcription. As illustrated in figure 3.4, CRE BEAS-2B cells were pretreated with vehicle, cilostazol (10 μM), GSK 256066 (10 nM), roflumilast N-oxide (10 nM), or zardaverine (10 µM) before stimulated with various concentrations of formoterol.
Cells were harvested after 6 h for luciferase assay. LogEC50 values and the fold activation were analysed by Student’s paired t-test.
56 3.1.2.5 Role of PKA in PDE inhibitor- and formoterol-induced, CRE-dependent transcription
To explore the potential role of PKA in the enhancement of CRE-dependent transcription by cAMP-elevating agents, an adenovirus vector, Ad5.CMV.PKIα, was used, which directs over-expression of PKIα, a highly selective PKA inhibitor (Meja et al., 2004). CRE BEAS-2B cells were infected with Ad5.CMV.PKIα, an empty adenoviral vector Ad5.CMV.Null (null) (control virus) or left untreated (naive). Cells were infected with the adenoviral vectors at a multiplicity of infection (MOI) of 30 as previous studies have demonstrated that this concentration of virus induced maximum expression of
PKIα in BEAS-2B cells (Meja et al., 2004). Then, concentration-response curves to formoterol were constructed in the absence and presence of siguazodan (10 µM) plus rolipram (10 µM). In naive cells or cells infected with Ad5.CMV.Null, formoterol alone increased luciferase activity with Emax = 17.4 ± 1.2-fold and 19.3 ± 1.7-fold, respectively.
The same effect was observed in naive cells and cells infected with Ad5.CMV.Null treated with formoterol plus PDE inhibitors (Emax = 21.8 ± 1.7 and 22.1 ± 1.9-fold, respectively) (Fig.3.5 A and B). In contrast, the ability of formoterol to induce luciferase activity was inhibited by more than 90% in cells infected with Ad5.CMV.PKIα. Similar inhibition was observed in cells stimulated with formoterol plus PDE inhibitors (Fig.3.5 A and B). Thus, these data imply that PKA plays the key role in the activation of CRE- dependent transcription by formoterol, whether in the absence or presence of combined
PDE3/PDE4 inhibition.
57
A B 25 30 Naive Naive 30 MOI Null 25 30 MOI Null 20 30 MOI PKI 30 MOI PKI 20 15 * * 15 * 10 * *
10 (fold induction) 5 CRE ReporterCRE Activity 5 N = 7 N = 7 0 0
NS -12 -11 -10 -9 -8
NS -12 -11 -10 -9 -8 R+S
(PKI) NS Log [Formoterol (M)]
NS (Null) NS Log [Formoterol (M)] PKI (R+S) PKI Null (R+S) Null +Sig&Roli
Figure 3.5. Effect of PKIα over-expression on CRE-dependent transcription. CRE BEAS-2B cells were left untreated (control) or infected at a multiplicity of infection (MOI) of 30 with either Ad5.CMV.PKIα or Ad5.CMV.Null for 48 h. Thereafter, cells were left untreated (A) or treated (B) for 0.5 h with siguazodan (Sig; 10 µM) plus rolipram (Roli; 10 µM), before being exposed to various concentrations of formoterol (A and B). After 6 h, cells were harvested to measure luciferase activity. Data, (n = 7) expressed as fold induction, are plotted as means ± SE. Statistical significance was determent using an ANOVA with a Bonferroni post-test is indicated where ***= P < 0.0001.
58 3.1.2.6 Formoterol promotes the phosphorylation of CREB
Several transcriptions factors can, theoretically, drive the activation of CRE-dependent transcription (Meja et al., 2004). CREB is one which has been shown to have a central role in many cells and tissues (Meja et al., 2004). To examine this hypothesis, western blots were used to determine if CREB is phosphorylated in a manner that is consistent with reporter activation. BEAS-2B CRE reporter cells were treated with a maximally- effective concentration of formoterol (10 nM) for 10 min, 30 min, 1 h, 2 h and 6 h.
Subsequently, the phosphorylation of CREB was analysed by western blotting (Fig.
3.6). Phosphorylation of CREB was readily detected at 10 min and was sustained for 1 h, after which CREB phosphorylation began to decline returning to basal levels at 6 h
(Fig. 3.6).
59
Figure 3.6. Effect of formoterol on CREB phosphorylation. BEAS-2B cells were treated with formoterol (10 nM) and harvested for protein after 1/6, 1/2, 1, 2 and 6 h. Cell lysates were subjected to western blot analysis for p-CREB, total CREB and GAPDH. Following densitometric analysis, data (n = 3), expressed as a ratio of pCREB/GAPDH, are plotted as means ± SE. Phosphorylation of CREB was analysed using ANOVA with a Dunnett's post-test is indicated; ** P < 0.01. (T. George, unpublished data).
60 3.1.2.7 PDE inhibitors enhance formoterol-induced, CREB phosphorylation
To examine the effect of PDE inhibitors on the activation of transcription factors (such as CREB and ATF-1), BEAS-2B cells were either not treated or pre-treated for 0.5 h with siguazodan (10 µM) and/or rolipram (10 µM), before stimulation or not with formoterol (10 pM or 100 pM) for 10 and 30 min. As is shown in figure 3.7, there was basal activation of phospho-CREB and phospho-ATF-1 in untreated cells. Although the lower concentration of formoterol (10 pM) alone very weakly activated pCREB/pATF-1 at 10 and 30 min, the higher concentration of formoterol (100 pM) clearly enhanced the phosphorylation of CREB and ATF-1 at 10 and 30 min (Fig. 3.7 A and B). Furthermore, the positive control, forskolin, induced a similar effect to formoterol (100 pM) (Fig. 3.7 A and B).
Alternatively, siguazodan and/or rolipram failed to alter basal phosphorylation of CREB and ATF-1 at any time point. Moreover, cells exposed to formoterol (10 pM) and siguazodan demonstrated increased, but not statistically significant, phosphorylation of
CREB and ATF-1 at 10 and 30 min (Fig. 3.7 A and B). Similarly, cells exposed to rolipram and formoterol (10 pM) induced significant phosphorylation of CREB and ATF-
1 at 10 min. However, the combination of siguazodan, rolipram and formoterol (10 pM) induced a significant increase in the phosphorylation of CREB and ATF-1 at 10 and 30 min. Alternatively, the addition of siguazodan and rolipram to cells treated with formoterol (100 pM) failed to induce difference in pCREB and pATF-1 level of activation at any time point compared to cells treated only with formoterol (100 pM). These data indicate that cAMP-elevating agents have partial effect on the transcription factors,
61 CREB and ATF-1, and maybe there are other transcription factors involved in the system.
Figure 3.7. Effect of siguazodan and rolipram on formoterol-induced pCREB and pATF-1. BEAS-2B cells were pre-incubated for 30 min with vehicle, or rolipram (Roli) and/or siguazodan (Sig) (both 10 μM). After 30 min, cells were treated with formoterol (10 or 100 pM) (Form). Cells were harvested after 10 and 30 min for protein and subject to western blot analysis for pCREB, pATF-1 and total CREB. Data (n = 4) expressed as a ratio of pCREB/CREB, are plotted as means ± SE. Expression of pCREB was analysed using ANOVA with a Bonferroni post-test is indicated where * = P < 0.05.
62 3.2 Aim 2: To investigate functional interactions between formoterol and PDE inhibitors on the induction of putative anti-inflammatory/protective genes in BEAS-2B cells.
3.2.1 Rationale
In the preceding section, data illustrate that the PDE inhibitors functionally interacted with the LABA, formoterol, to promote luciferase expression in 6xCRE BEAS-2B reporter cells. The next step in the investigation attempted to ascertain if this effect applies to putative anti-inflammatory/protective genes.
In a related project, BEAS-2B cells were stimulated with LABA and PDE4 inhibitor alone and in combination prior to genome-wide microarray analysis. Genes whose expression was enhanced by these treatments and which showed potential anti-inflammatory properties were selected and examined for their ability to be regulated by LABA and
PDE inhibitors. The genes selected were: regulator of G-protein signalling 2 (RGS2), mitogen-activated protein (MAP) kinase phosphatase (MKP-1), suppressor of cytokine signaling 3 (SOCS3), cysteine-rich secretory protein LCCL (Limulus clotting factor C,
Cochlin, Lgl1) domain-containing 2 (CRISPLD2), and cluster of differentiation 200
(CD200). Brief details of their putative functions are provided:-
RGS2: Gq is a class of heterotrimeric G proteins that link a variety of GPCRs to the mobilization of Ca2+ (Hubbard and Hepler, 2006). In the airways, these receptors mediate many of the key effects of inflammatory mediators, including histamine and leukotrienes, which produce bronchoconstriction and may promote mediator release from epithelial cells (Barnes, 2008). The termination of this Gq-regulated signalling occurs in part through the activation of GTPase-activating proteins, such as RGS2.
63 RGS2 promotes the hydrolysis of GTP to GDP and, thereby, returns the Gαq to its inactive form (Heximer, 2004; Kimple et al., 2009). According to Holden et al., (2011), the LABA, salmeterol, particularly in the presence of glucocorticoid, induces the expression of RGS2 via the classical cAMP-PKA cascade.
CD200: CD200 also called OX-2, it is a highly glycosylated membrane protein expressed on the cell surface in various tissues. CD200 act as a negative regulator of airway macrophages by binding to macrophage CD200R receptors and preventing their activation (Snelgrove et al., 2008). Studies in mice have revealed that macrophage activation was impaired when CD200 bind to CD200R causing a decrease in pro- inflammatory cytokine production (Snelgrove et al., 2008). Acute exacerbations of
COPD are mainly stimulated by extended periods of excessive inflammation that often represent the response to bacterial and/or viral infections. Thus, upregulation of CD200 on airway cells in COPD patients could mitigate inflammation and decrease the frequency of exacerbations (Moodley et al., 2013).
CRISPLD2: Recent studies found that CRISPLD2 functions as a novel, secreted, mammalian lipopolysaccharide (LPS)-binding protein in humans and mice. CRISPLD2 acts as a natural LPS antagonist and has the ability to inhibit endotoxic shock (Wang et al., 2009). Increased CRISPLD2 expression may support decreases in COPD exacerbations produced by gram-negative bacterial infections. CRISPLD2 can bind to the LPS found on the surface of gram-negative bacteria and reduce the activation Toll- like receptor 4 (TLR4)-mediated pro-inflammatory responses (Moodley et al., 2013;
Wang et al., 2009).
64 SOCS3: Suppressor of Cytokine Signalling 3, which, as suggested by its name, is involved in negative regulation of cytokine signalling mediated by the JAK-STAT pathway (Yasukawa et al., 1999) . SOCS3 negatively regulates cytokines such as IFNγ, which is involved in the production of chemo-attractant molecules that induce the recruitment of Th1 cells and neutrophils in the lung (Song and Shuai, 1998). Thus, increasing SOCS3 expression could inhibit pulmonary leukocytes recruitment and, therefore, inflammatory responses in the lung (Gao et al., 2006).
MKP-1: MKP-1 is an important negative regulator of all MAPK pathways (Clark et al.,
2008). MAP kinases fulfill multiple functions in inflammation, cellular proliferation, differentiation and to some extent ASM contractile responses (Johnson and Lapadat,
2002; Quante et al., 2008; Rincón and Davis, 2009). MKP-1 acts by dephosphorylating the tyrosine and threonine residues that are necessary for MAPK activation (Boutros et al., 2008; Liu et al., 2007).
3.2.2 Results
3.2.2.1 Effect of formoterol and PDE inhibitors on the expression of anti- inflammatory/protective genes
To examine the interaction between formoterol and PDE inhibitors on the induction of anti-inflammatory/protective genes, BEAS-2B cells were either not stimulated or stimulated with maximally-effective concentrations of siguazodan (10 μM) plus rolipram
(10 μM) for 30 min, prior to stimulation with formoterol (1 nM). Cells were harvested at
1, 2, 6, 12, and 18 h for real-time PCR analysis of RGS2, MKP-1, SOCS3, CRISPLD2,
CD200 and, as a normalisation control, GAPDH. Formoterol induced RGS2 expression maximally at 1 h, while siguazodan plus rolipram were very weak activators of RGS2
65 (less than 3-fold induction). The combination formoterol/siguazodan plus rolipram did not show additive or synergetic effects at 1 or 2 h (Fig. 3.8 A). Thereafter, RGS2 expression decreased with time. Of note, was the fact that in cells treated with the combination of formoterol, siguazodan and rolipram, RGS2 mRNA did not decrease as rapidly as in those cells exposed to formoterol only.
In cells treated with formoterol, the expression of CD200 was induced significantly by
4.3 fold at 1 h and by 5.8 fold at 2 h. However, the expression waned at 6, 12 and 18 h.
Siguazodan plus rolipram did not significantly alter the basal expression of CD200 at any time point. Moreover, cells treated with a combination of siguazodan, rolipram and formoterol did not result in further increase in CD200 mRNA expression compared to cells treated with formoterol alone at 1, 2 and 6 h (Fig. 3.8.B). Similar to RGS2 expression, the presence of siguazodan plus rolipram in cells treated with formoterol modestly enhanced the expression of CD200 at 12 and 18 h.
Formoterol did not induce CRISPLD2 or SOCS3 at 1 h; however, after 2 h of treatment with formoterol, the mRNA of CRISPLD2 and SOCS3 were increased by 18- and 2.6- fold respectively. By 6 h, this effect had waned dramatically. On the other hand, siguazodan plus rolipram did not induce these genes at any of the time points (Fig. 3.8
C and E).
MKP-1 was also screened. As shown in figure 3.8 D, cells treated with formoterol maximally expressed MKP-1 at 1 h and this expression decreased gradually thereafter.
The additional presence of siguazodan plus rolipram failed to markedly modulate the expression of MKP-1 at any time point.
66
A 15 * * * *
10 D * * * * 6 * * * *** *** *** *
5 * * Fold (NS 1h) (NS Fold
RGS2/GAPDH * * * * * * * * * * * 4 0 B
8 2
Fold (NS 1h) (NS Fold MKP-1/GAPDH * * * * 6 * * 0 * * * 4 * E * * *** ** 4 * * * * Fold (NS 1h) (NS Fold * * CD200/GAPDH 2 * * * 3 * 0 C 2 25 * * * 1h) (NS Fold 1 * * SOCS3/GAPDH 20 *
15 0 Roli+Sig + + + + + + + + + + Form + + + + + + + + + + 10 * Time (h) 1 2 6 12 18 * * Fold (NS 1h) (NS Fold * *
5 * CRISPLD2/GAPDH
0 Roli+Sig + + + + + + + + + + Form + + + + + + + + + + Time (h) 1 2 6 12 18
Figure 3.8. Effect of LABA and PDE inhibitors on CD200, CRISPLD2, RGS2, MKP-1 and SOCS3 gene expression. BEAS-2B cells were pre-incubated for 30 min with vehicle, or a combination rolipram and siguazodan (both 10 μM) (Sig + Roli) and then exposed to formoterol (1 nM). Cells were harvested after 1, 2, 6, 12 and 18 h for real time RT-PCR. Data (n = 4) were normalized to GAPDH, expressed as fold change over non-stimulated (NS) cells at 1 h and plotted as mean ± SE. Significance, relative to NS at each time point, using an ANOVA with a Bonferroni post-test is indicated where * = P < 0.05, ** = P < 0.01 and *** = P < 0.001. Additional comparisons are as indicated.
67 3.2.2.2 PDE inhibitors sensitise BEAS-2B cells to formoterol-induced gene induction
As illustrated by the reporter data (Fig. 3.3), PDE inhibitors with high concentrations of formoterol failed to increase luciferase activity more than formoterol alone. In contrast, the presence of PDE inhibitors with lower concentrations of formoterol showed significant sensitisation. Thus, lower concentrations of formoterol functionally interacted with PDE inhibitors to enhance gene expression.
BEAS-2B cells were either not treated or treated with the maximum effective concentrations of siguazodan (10 μM) and/or rolipram (10 μM) for 30 min, prior to stimulation with increasing concentration of formoterol. The cells were harvested at 2 h, for real-time PCR analysis, to allow sufficient time for the maximal induction of gene expression.
The results demonstrated that neither siguazodan nor rolipram were independently able to induce significant expression of RGS2, CD200 or CRISPLD2, relative to untreated cells. However, treatment involving both siguazodan plus rolipram demonstrated significant increases of RGS2, CD200, and CRISPLD2 expression (Fig. 3.9 A-C) by 2.2-
, 3.6- and 3.6-fold respectively (P < 0.001). Formoterol alone induced the expression of
RGS2, CD200, and CRISPLD2 in a concentration dependent manner. In the presence of siguazodan plus rolipram, the expression RGS2, CD200, and CRISPLD2 were significantly enhanced at the lower concentrations of formoterol. For instance, the expression of RGS2 mRNA induced by 1 or 10 pM of formoterol was increased from
1.4- to 3.5-fold and from 2.8- to 6-fold respectively in the presence of siguazodan plus rolipram (Fig. 3.9 D).
68 Similar effects were seen with CRISPLD2 and CD200 mRNAs (Fig 3.9 E and F).
Specifically, formoterol induced CRISPLD2 expression was increased from 1.5- to 4.8- fold, from 3.7- to 10.4-fold, and from 10.7- to 12.7-fold at 1, 10 and 100 pM respectively in the presence of siguazodan plus rolipram. While formoterol induced CD200 expression was enhanced from 1.7- to 5.4-fold and from 4.2- to 9-fold at 1 and 10 pM respectively in the presence of siguazodan plus rolipram. Moreover, siguazodan plus rolipram decreased the apparent logEC50 of formoterol response curve for RGS2,
CRISPLD2 and CD200, from -10.8 to -11.8, from -10.7 to -11.4 and from -10.9 to -11.6 respectively (Fig 3.9 D-F). These data demonstrate that PDE inhibitors have the ability to sensitise BEAS-2B cells to the expression of formoterol-inducible, anti- inflammatory/protective genes.
69 A B C 4 ** 8 *** * 6 *** ** 3 *** 6 *** *** *** 4 2 4 ns ns ns ns
RGS2/GAPDH 2
CD200/GAPDH
(fold induction) (fold induction) 1 (fold induction) 2 ns
CRISPLD2/GAPDH ns
0 0 0 Sig + + Sig + + Sig + + Roli + + Roli + + Roli + + D E F 8 15 15 Naive ** Naive *** Naive Roli+Sig Roli+Sig *** Roli+Sig 6 10 10 **
4 ** ** ***
5 5
RGS2/GAPDH
CD200/GAPDH
(fold induction) (fold induction) (fold
(fold induction) (fold 2 CRISPLD2/GAPDH N= 6 N= 6 N= 6 0 0 0
NS -12 -11 -10 -9 NS -12 -11 -10 -9 NS -12 -11 -10 -9 Log [Formoterol (M)] Log [Formoterol (M)] Log [Formoterol (M)] Roli+Sig Roli+Sig Roli+Sig
Figure 3.9. Effect of inhibitors of PDE 3 and PDE 4 on formoterol-inducible genes. BEAS-2B cells were incubated for 30 min with rolipram (10 µM) (Roli) and/or siguazodan (10 µM) (Sig), before simulation with various concentrations of formoterol (Form). After 2 h, cells were harvested for mRNA and real-time PCR was performed for RGS2, CRISPLD2, CD200 and GAPDH. Data (n = 5) are normalized to GAPDH and expressed as fold change over non-stimulated (NS) at 2 h. Significance, relative to NS, using an ANOVA with a Bonferroni post-test is indicated where * = P < 0.05, ** = P < 0.01 and *** = P < 0.001, and additional comparisons are as indicated, in panel A, B and C. Alternatively, a Student’s paired t-test was used to determine significant of the data in panel D, E and F, where ** = P < 0.01 and *** = P < 0.001.
70 3.3 Aim 3: To examine the effect of PDE inhibitors and formoterol on the expression of glucocorticoid-inducible anti-inflammatory/protective genes in BEAS-2B cells
3.3.1 Rationale
The ability of glucocorticoids to increase β2-adrenoceptor mediated signalling is well known (Barnes, 2002; Giembycz and Newton, 2006). However, studies have not yet clarified the way in which β2-adrenoceptor agonists increase glucocorticoid signalling responses (Giembycz and Newton, 2006). The current body of literature shows that the stimulation of anti-inflammatory effector genes fulfills an important function in the anti- inflammatory effects of glucocorticoids (Abraham and Clark, 2006; Newton, 2000).
Moreover, there are data showing that β2-adrenoceptor agonists, and other cAMP- elevating agents, have the ability to augment glucocorticoid anti-inflammatory genes expression (Kaur et al., 2008). Accordingly, the hypothesis of this study maintains that the addition of other cAMP-elevating agents, such as inhibitors of PDE3 and PDE4, can induce, further enhance or prolong the induction of glucocorticoid-induce anti- inflammatory genes. Thus, the next step of the current investigation involves examining the effect of PDE inhibitors in the absence or presence of LABA on glucocorticoid- induced genes. The same genes that were selected to examine the interaction between
LABA and PDE inhibitors in CRE reporter cells were used in these studies.
3.3.2 Results
3.3.2.1 Effect of dexamethasone on the expression of anti-inflammatory/protective genes in combination with cAMP-elevating agents
To investigate the effect of cAMP elevating agents (formoterol, siguazodan and rolipram) on the induction of anti-inflammatory/protective genes with glucocorticoid,
71 BEAS-2B cells were left untreated or were pre-treated for 30 min with siguazodan plus rolipram each at 10 µM. Subsequently, cells were stimulated with 1 µM dexamethasone in the absence or presence of 1 nM of formoterol. Cells were then harvested after 1, 2,
6, 12, and 18 h for real-time PCR analysis.
Dexamethasone weakly induced RGS2 mRNA expression at each time point (Fig. 3.10
A). Conversely, the addition of siguazodan plus rolipram enhanced this induction slightly from 2.3- to 6.3-fold, 3.3- to 7.3-fold, 3.8- to 7.2-fold, 3.2- to 5.7-fold and 2- to 3.5-fold at
1, 2, 6, 12 and 18 h respectively. When cells were treated with dexamethasone plus formoterol, RGS2 mRNA levels were maximal at 1 h, with a 36.6-fold enhancement over untreated cells (Fig. 3.10 A), and a 12.2-fold increase over cells treated only with formoterol (Fig. 3.8 A).
At the early time points (1 and 2 h), the prior addition of siguazodan plus rolipram failed to increase the maximum level of RGS2 mRNA induced by dexamethasone plus formoterol alone. However, an effect of the PDE inhibitors was observed at late time points, where siguazodan plus rolipram markedly reduced the rate of decline in RGS2 mRNA. Thus, at 6, 12 and 18 h the expression levels were 24.2-, 12.1- and 7.9-fold respectively relative to unstimulated cells, whereas in cells treated only with dexamethasone and formoterol expression levels were correspondingly 16.6-, 5.6- and
3.5-fold (Fig. 3.10 A).
Dexamethasone induced very weak expression of CD200 at 12 and 18 h (1.8- and 2.2- fold respectively). The addition of siguazodan plus rolipram increased CD200 expression weakly at 1 and 2 h (2.7- and 2.8-fold respectively). The combination of
72 formoterol and dexamethasone induced a synergistically CD200 expression. Thus, formoterol alone induced CD200 by 4.3-fold at 1 h and by 5.8-fold at 2 h (Fig. 3.8 B), while formoterol and dexamethasone together enhanced the expression of CD200 at 1 and 2 h by 7.3- and 11.6-fold respectively (Fig. 3.10 B). After 2 h, the expression of
CD200 mRNA decreased. Although, the induction of CD200 by dexamethasone and formoterol was unaffected by siguazodan plus rolipram at early time points (1, 2 and 6 h), their addition reduced the rate of decline of CD200 mRNA observed by 12 and 18 h
(Fig. 3.10 B).
Alone, dexamethasone induced CRISPLD2 mRNA expression by 7.5- and 9.5-fold at 2 and 6 h respectively and the addition of siguazodan plus rolipram enhanced this to 11- and 15-fold respectively (Fig. 3.10 C). Formoterol alone also induced by 18.9- and 5.7- fold the expression of CRISPLD2 at 2 and 6 h respectively (Fig. 3.8 C). The addition of formoterol to cells treated with dexamethasone induced a significant induced in
CRISPLD2 mRNA by 50.3- and 34.3-fold at 2 and 6 h respectively (Fig. 3.10 C).
Although, the induction of CRISPLD2 mRNA by dexamethasone and formoterol was unaffected by siguazodan/rolipram at 1, 2, 6 and 12 h, they enhanced slightly the relative induction at 18 h (Fig. 3.10 C).
MKP-1 expression was induced at 1 h by dexamethasone (4.8-fold) and the addition of siguazodan/rolipram had no further effect (5.5-fold). While formoterol alone was able to induce the expression of MKP-1 mRNA by 4.8-fold at 1h (Fig. 3.8 D), the addition of formoterol to cells exposed to dexamethasone augmented this induction to 9.3-fold.
After 1 h the expression of MKP-1 mRNA declined and all treatments alone or in combination induced approximately the same level of induction (Fig. 3.10 D).
73 Unlike the other genes examined, dexamethasone inhibited the expression of SOCS3 at all-time points, and none of the treatments reversed this effect (Fig. 3.10 E).
These data illustrate that PDE inhibitors have the ability to prolong the expression of some LABA/glucocorticoid inducible genes.
74 A * * 40 * * * * * *** * 30 * * * * 20 * *** 15 * D * *** 15
10 * Fold (NS 1h) (NS Fold RGS2/GAPDH * * * * * * 5 * * * * * * * 10 * 0 * B * * * * * 5 * * 15 * * * * 1h) (NS Fold * * * * * * * * * * * * MKP-1/GAPDH * * * * * * * * * * * * * * * * * * *
* 10 * 0 * * * * E *** *** 5 * * 5 * * * * Fold (NS 1h) (NS Fold * * * CD200/GAPDH * * * * * * * * * 4 * * * * * * 0 3
C 2 * 60 * 1h) (NS Fold * SOCS3/GAPDH 1 * 40 * * * * 0 Dex + + + + + + + + + + + + + + + + + + + + 20 * * Roli+Sig + + + + + + + + + + 15 Form + + + + + + + + + + Time (h) 1 2 6 12 18 10 * * ** Fold (NS 1h) (NS Fold *** * * * * * * * * * * * * CRISPLD2/GAPDH * 5 * * * * * * * 0 Dex + + + + + + + + + + + + + + + + + + + + Roli+Sig + + + + + + + + + + Form + + + + + + + + + + Time (h) 1 2 6 12 18
Figure 3.10. Effect of siguazodan, rolipram and formoterol in the presence of dexamethasone on the expression of genes. BEAS-2B cells were pre-treated with siguazodan (10 µM) (Sig) plus rolipram (10 μM) (Roli) for 30 min, before simulation with formoterol (1 nM) (Form) and dexamethasone (1 µM) (Dex). Cells were harvested at 1, 2, 6, 12 and 18 h and RNA was extracted. Following cDNA synthesis, SYBR green real- time PCR was performed for RGS2 (A), CD200 (B), CRISPLD2 (C), MKP-1 (D) and SOCS3 (E). Data (n = 4), normalized to GAPDH and expressed as fold increase over non-stimulated cells (NS) at 1h, are plotted as means ± SE. Significance, relative to NS at each time point, using an ANOVA with a Bonferroni post-test is indicated where * = P < 0.05, ** = P < 0.01 and *** = P < 0.001. Additional comparisons are as indicated.
75 3.3.2.2 PDE inhibitors prolong the expression of RGS2 mRNA
To more clearly assess the effect of PDE inhibitors on prolonging the expression of genes, the RGS2 data were re-plotted. As seen in figure 3.11, cells treated with dexamethasone plus formoterol as well as those treated with dexamethasone plus formoterol and siguazodan plus rolipram induced a similar maximal effect on the expression of RGS2 mRNA at 1 h. In each case, the RGS2 mRNA expression gradually declined after 1 h. While the effect of both of the combination treatments decreased overtime, the expression of RGS2 in cells treated with dexamethasone plus formoterol alone declined faster than those treated with a combination of dexamethasone, formoterol and siguazodan plus rolipram. As shown in figure 3.11 A and B, in cells treated with dexamethasone plus formoterol, RGS2 mRNA expression declined to 50 or
25% of the maximum (i.e. 100% at 1 h) by ~5.3 and ~9.3 h respectively, whereas in cells treated with dexamethasone, formoterol and siguazodan/rolipram this was prolonged to ~8 and ~15.9 h respectively. These data therefore illustrate a functional interaction between LABAs plus glucocorticoids and PDE inhibitors.
76
100 Form+Dex 100 Form+Dex Form+Dex+Roli+Sig Form+Dex+Roli+Sig
75 * 75 * (%) (%) 50 50
** **
RGS2/ GAPDH RGS2/ GAPDH 25 ** 25 **
N=4 N=4 0 0 1 2 6 12 18 1 2 6 12 18 Time (h) Time (h)
~5.3 h ~8 h ~9.3 h ~15.9 h
Figure 3.11. Effect of PDE inhibitors on RGS2 expression. Data from figure 3.10 C were re-plotted and the effect of combination drugs is expressed as a percentage. 100% indicates expression of RGS2 at 1 h. The curve with ( ) represent cells treated with dexamethasone (1 µM) (Dex) and formoterol (1 nM) (Form). While the curve with ( ) represent cells treated with dexamethasone (1 µM), formoterol (1 nM) (Form), siguazodan (10 µM) (Sig) and rolipram (10 µM) (Roli). Data (n = 4) are plotted as means ± SE. Statistical significance was performed using a Student’s paired t-test, where * = P < 0.05 and ** = P < 0.01.
77 3.3.2.3 PDE inhibitors sensitise BEAS-2B cells to LABA/glucocorticoid-induced gene induction
Given the ability of PDE inhibitors to sensitise but not enhance the maximum response of the reporter system, we sought to utilize a lower concentration of LABA to show functional interactions between inhibitors of PDEs, LABA and glucocorticoid.
Therefore, to examine a role for PDE inhibitors in sensitising BEAS-2B cells to
LABA/glucocorticoid induced gene induction, cells were pre-treated with siguazodan (10
µM) and rolipram (10 µM), before being stimulated with a fixed concentration of dexamethasone (1 µM) in the absence or presence of increasing concentrations of formoterol for 2 h.
Siguazodan plus rolipram enhanced the expression of RGS2, CRISPLD2 and CD200 induced by dexamethasone significantly (from 2.7- to 9.2-fold, 3.3- to 7.4-fold, 2.4-to
7.4-fold respectively) (Fig. 3.12 A-C).
Formoterol and dexamethasone enhanced the expression of RGS2, CRISPLD2 and
CD200 in a concentration-dependent manner (Fig. 3.12 D-F). Furthermore, siguazodan plus rolipram significantly enhanced dexamethasone/ formoterol-inducible genes
(RGS2, CRISPLD2 and CD200) at the lower concentrations of formoterol. For example,
RGS2 mRNA expression induced by a fixed concentration of dexamethasone (1 µM) and 1, 10 or 100 pM of formoterol was enhanced from 6- to 20.8-fold, from 15.8- to
36.8-fold and from 35.2- to 43.1-fold respectively in the presence of siguazodan plus rolipram. Moreover, the logEC50 values of the dexamethasone/formoterol response curve that describe RGS2 induction was decreased in the presence of siguazodan plus rolipram (from -10.75 to -11.84) (Fig. 3.12 D).
78 Alternatively, the expression of CRISPLD2 by 1 µM dexamethasone and 1 or 10 pM of formoterol was enhanced by siguazodan plus rolipram, from 5.4- to 11.9-fold and from
12.7- to 21.1-fold respectively (Fig. 3.12 E). Moreover, a similar effect was seen on
CD200 expression (Fig. 3.12 F).
These results show that inhibitors of PDE3 plus PDE4 were able to induce significant enhancements in the expression of anti-inflammatory/protective genes in the presence of glucocorticoid. Furthermore, PDE inhibitors have the ability to sensitise BEAS-2B cells to the gene inducing activity of LABA and glucocorticoid in combination.
79 A B C 15 15 10 *** *** *** ** 8 *** 10 10 *** 6
4
5 5 ns CD200/GAPDH
(foldinduction) *
RGS2/GAPDH (foldinduction)
* (fold induction) 2 CRISPLD2/GAPDH
0 0 0 Dex + + Dex + + Dex + + Roli+Sig + Roli+Sig + Roli+Sig +
D E F 40 40 50 Dex ** Dex Dex Dex+Roli+Sig Dex+Roli+Sig Dex+Roli+Sig 40 ** 30 30 * ** 30 ** 20 20
20 ** *** (fold induction) (fold
10 CD200/GAPDH 10 (fold induction) (fold
RGS2/GAPDH 10
CRISPLD2/GAPDH induction) (fold N= 5 N= 5 N= 5 0 0 0 -12 -11 -10 -9 -12 -11 -10 -9 -12 -11 -10 -9 Dex Dex Dex Log [Formoterol (M)] Log [Formoterol (M)] Log [Formoterol (M)]
Dex+Roli+Sig Dex+Roli+Sig Dex+Roli+Sig
Figure 3.12. Effect of siguazodan and rolipram on formoterol+dexamethasone- inducible gene expression. BEAS-2B cells were pretreated or not for 30 min with rolipram (10 µM) (Roli) and/or siguazodan (10 µM) (Sig), before stimulation with dexamethasone (1 µM) (Dex) alone or combined with various concentrations of formoterol (Form). Cells were harvested after 2 h for mRNA and SYBR green real-time PCR was performed for RGS2, CRISPLD2 and CD200. Data (n = 5), normalized to GAPDH and expressed as fold over non-stimulated (NS) at 2 h, are plotted as means ± SE. Significance, relative to NS, using an ANOVA with a Bonferroni post-test is indicated where * = P < 0.05 and *** = P < 0.001 and additional comparisons are as indicated, in panel A, B and C. Alternatively, a Student’s paired t-test was used to determine significant of the data in panel D, E and F, where * = P < 0.05, ** = P < 0.01 and *** = P < 0.001.
80 Chapter four: Discussion
A growing body of literature suggests that the core anti-inflammatory actions of glucocorticoids, i.e. the repression of inflammatory gene expression, may result from the repression of inflammatory transcription factors as well as the transactivation of many anti-inflammatory genes (Newton and Holden, 2007; Newton et al., 2010). Similarly, it is possible that PDE inhibitors acting alone or in combination with LABAs may provide positive therapeutic effects in COPD via transcriptional activation of cAMP-activated factors such as CREB. Because PDE inhibitors and LABAs both enhance cAMP, the combination of such medications may lead to further increased levels of cAMP.
Moreover, clinical studies have demonstrated the greater therapeutic benefits of combining a LABA, which elevates cAMP, and an ICS (Shrewsbury et al., 2000;
Woolcock et al., 1996). Given this, it is reasonable to speculate that the addition of multible cAMP-elevating agents, such as inhibitors of PDE3 and PDE4, may enhance further the therapeutic benefits of combining LABA and ICS.
The molecular mechanism of action for the enhanced therapeutic benefit of combining a cAMP-elevating agent (e.g. a LABA) and an ICS has not yet been clarified. Data show that the transcription of some genes induced by glucocorticoids may be enhanced by cAMP-elevating agents (Newton et al., 2010). Accordingly, this thesis proposes that the use of different cAMP-elevating agents can interact mechanistically to activate the classical cAMP/PKA/CREB signalling pathway to promote CRE-dependent transcription. This may also lead to the enhancement and/or prolongation of the expression of anti-inflammatory and/or protective genes. Moreover, the addition of
81 glucocorticoids to those cAMP-elevating agents may enhance further the expression of anti-inflammatory and/or protective genes.
4.1 Effect of PDE inhibitors on CRE-dependent transcription
Treatment with individual PDE inhibitors failed to markedly stimulate luciferase activity in 6xCRE BEAS-2B reporter cells (Fig. 3.1). These results concur with data in mesangial cells, where neither PDE3 inhibitors nor PDE4 inhibitors, independently stimulated CRE activation (Zhu et al., 2006). Given the presence of both PDE3 and
PDE4 in these cells and BEAS-2B cells (Dent et al., 1998; Fuhrmann et al., 1999; Zhu et al., 2006), these findings may indicate that there is a low baseline synthesis of cAMP that is not sufficient to allow PDE inhibitors to enhance cAMP to a level necessary for obtaining a marked response on CRE-dependent transcription. However, both siguazodan and rolipram independently caused modest, but significant, leftward displacements towards higher potency of the concentration-response curves that described formoterol-induced CRE-dependent transcription (Fig.3.2 B and C).
Moreover, when siguazodan and rolipram were used in combination, a significantly greater displacement of the formoterol concentration-response curve was produced (log
EC50 values: from -9.9 to -10.8) (Fig.3.3 D). These data show a clear interaction between the different cAMP-elevating agents and indicate that, therapeutically, they could act together to produce added clinical benefit. In Cazzola et al. (2001), they constructed a dose response curve to formoterol, and showed that 18 µg was the maximum effective strength of the drug to patients with mild acute exacerbation of
COPD (Cazzola et al., 2001). Alternatively, the recommended dose of formoterol to treat COPD patients, according to the GOLD, is between 4.5-12 µg
82 (http://www.goldcopd.org/uploads/users/files/GOLD_Report_2013_Feb20.pdf). Thus, the recommended doses of formoterol are likely to represent sub-maximal effective concentrations. From this, it can be speculated that the addition of inhibitors of PDE3 and PDE4 may enhance the therapeutic effect of formoterol in COPD patients.
4.2 Effect of PDE inhibitors on gene expression
Based on the findings from the CRE reporter experiments, we sought to explore the effect of PDE inhibition on the enhancement of gene expression by LABA and/or glucocorticoid. Prior microarray profiling in different types of airway epithelial cells identified several potential genes that were induced by dexamethasone or LABA and
PDE4 inhibitors respectively. Genes such as RGS2, SOCS3, MKP-1, CD200 and
CRISPLD2 were selected for the current analysis based on the fact that they may provide clinical benefits in COPD treatment.
Siguazodan and rolipram together exerted a small yet significant effect on gene induction (Fig. 3.9 A-C). The low level of gene expression may be of particular relevance in cells such as airway epithelia, where the spontaneous rate of cAMP formation in BEAS-2B cells is low. Thus, the effect of siguazodan and rolipram to prevent the degradation of cAMP needed to induce gene expression will be low.
Alternatively, the effect of genes induced by inhibitors of PDE3 and PDE4 can be higher in other cells where the level of cAMP is high. For example, in airway myocytes, which have been suggested to have a high level of cAMP (Lazzeri et al., 2001), one can predict that inhibitors of PDE3 and PDE4 will induce higher expression of genes.
Moreover, in vivo, cAMP-generating ligands, such as prostaglandin E2 (PGE2), can be
83 released from many diverse cell types (Wilson et al., 2011) as well as via the therapeutic administration of β2-adrenoceptor agonists.
The LABA, formoterol, produced maximum stimulation of RGS2, CRISPLD2, CD200,
MKP-1 and SOCS3 mRNA expression during the early time periods (1 and 2 h) (Fig.
3.8). While combination of siguazodan plus rolipram failed to enhance this effect, they managed to attenuate/reduce the rapid decrease in RGS2 and CD200 expression observed at later time periods (12 and 18 h) (Fig. 3.8 A and B). Furthermore, treatment of cells with dexamethasone and formoterol in combination yielded synergistic increases in the expression of RGS2, CRISPLD2 and CD200, and an additive effect on the expression MKP-1 at early time points (1 and 2 h) (Fig. 3.10). Again, siguazodan plus rolipram failed to produce further increases in the expression of these genes at these points. However, these drugs again resulted in a sustained expression of RGS2 and CD200 at the late time points (6, 12 and 18 h) (Fig. 3.10 A and B).
The inability of PDE inhibitors to enhance gene expression during early time periods was consistent with the reporter data, which demonstrated that siguazodan plus rolipram did not augment the maximum effect of high concentrations of formoterol (Fig.
3.3 D). These findings may be a consequence of using BEAS-2B cells (a model of human airway epithelial cell), which contain a relatively high number of β2- adrenoceptors approximately 4000 to 8000 per cell; (Johnson, 2002) allowing formoterol to produce the maximum response for each of these outputs (Fig. 3.8 and 3.10).
However, when low, sub-maximal, concentrations of formoterol were used, siguazodan plus rolipram were able to enhance the expression of genes significantly (Fig. 3.9 and
3.12). This shows that siguazodan plus rolipram were able to sensitise BEAS-2B cells to
84 the induction of gene expression produced by formoterol. Thus, the effect of inhibitors of
PDE3 and PDE4 may be more beneficial in different type of cell types where the number of β2-adrenoceptors is low. For example, T-lymphocytes, which are implicated in the pathogenesis of COPD, have been found to co-express PDE3 and PDE4 (Banner and Press, 2009). In addition, those cells have a low number of β2-adrenoceptors (~750 per cell) (Johnson, 2002). In this type of cell, it can be expected that activation of signalling by maximally-effective concentrations of formoterol may only produce partial responses on account of the low number of β2-adrenoceptors. Moreover, since the expression of formoterol-inducible genes in this type of cell may not reach the maximum due to the low signalling, the addition of siguazodan and rolipram can enhance the expression of formoterol-inducible genes highly.
Whilst formoterol stimulated the expression of SOCS3, PDE inhibitors neither increased nor sustained SOCS3 mRNA (Fig. 3.8 E) and conversely, glucocorticoids completely suppressed this expression (Fig. 3.10 E). While this phenomenon currently lacks a clear explanation, other investigators have demonstrated that glucocorticoids can also negatively regulate cAMP-inducible genes. For example, a study by Akerblom et al.,
(1988) showed that the glucocorticoid, dexamethasone, inhibited the expression of the glycoprotein hormone alpha-subunit gene. Although, glucocorticoids inhibit the induction of the anti-inflammatory gene SOCS3, this effect may be less important clinically. This is due to the fact that the clinical data have demonstrated the superior efficacy of combining cAMP-elevating agents and glucocorticoids (see Shrewsbury et al., 2000;
Woolcock et al., 1996), which may be explained by their additive or synergistic effects on gene transactivation (Newton et al., 2010).
85 4.3 The interaction between PDE3 and PDE4 inhibitors
Several PDE family members are expressed in structural and pro-inflammatory cells, and the combined targeting of these isoenzymes with multiple inhibitors would, theoretically, exert greater anti-inflammatory activity than that of a PDE4 inhibitor alone
(Giembycz and Newton, 2011). In vitro, research has demonstrated that the selective inhibition of PDE3 in T-lymphocytes, monocytes, and alveolar macrophages increases the effectiveness of a PDE4 inhibitor (Banner and Press, 2009). For example, many of the existing studies provide evidence that PDE3 and PDE4 inhibitors effectively reduce inflammation by suppressing the LPS-induced release of TNFα in inflammatory cells
(Gantner et al., 1999; Jin and Conti, 2002; Schudt et al., 1993). Specifically, investigations have found that PDE4 inhibitors exert a slight inhibitory effect on LPS- induced TNFα in alveolar macrophages (Schudt et al., 1993). Whereas, a dual PDE3/4 inhibitor fully suppresses the release of pro-inflammatory mediators (Schudt et al.,
1993). Moreover, the small airways of COPD patients have demonstrated a substantial increase in dendritic cells, exhibiting a positive association between dendritic cell influx and disease severity (Gantner et al., 1999). Such cells have been shown to release
TNFα upon stimulation by LPS. Whereas the PDE3 inhibitor, motapizone, only exhibited a small inhibitory effect alone, PDE4 inhibitor, rolipram, was able to suppress TNFα by almost 30%. Alternatively, the combination of rolipram and motapizone synergistically decreased TNFα output in dendritic cells (Gantner et al., 1999). As is the case with macrophages, these results indicate that PDE3 plus PDE4 inhibitors are required to fully inhibit the pro-inflammatory mediator release from dendritic cells (Banner and Press,
2009). As stated above, it is possible that the suppression of inflammation may involve
86 transactivation of anti-inflammatory genes. Our investigations demonstrate that PDE3 plus PDE4 inhibitors significantly stimulated the expression of CRISPLD2 mRNA (Fig.
3.9 D). This gene encodes a LPS-binding protein and, therefore, may act to reduce pro- inflammatory signalling through TLR4, an LPS receptor, in inflammatory cells (Wang et al., 2009). Therefore, it is possible that CRISPLD2 negatively regulates the expression of TNFα from pro-inflammatory cells in response to LPS.
4.4 Interaction between PDE inhibitors and LABA
In addition to countering pro-inflammatory effects, PDE4 inhibitors likely constitute a useful strategy for enhancing the therapeutic effect of other anti-inflammatory drugs.
This method could be especially apparent in respect of ICSs, which provide effective therapy for treating inflammation in asthma, but remain relatively ineffective in treating
COPD (Giembycz and Newton, 2011). Accordingly, patients with mild COPD are frequently managed with LABAs and SABAs on an as-needed basis (O'Donnell et al.,
2008). These medications, which provide purely symptomatic relief, work mainly through the cAMP/PKA pathway, indicating that PDE4 inhibitors have the potential to sensitise and possibly enhance β2-adrenoceptor-mediated effects. Nevertheless, since
PDE4 inhibitors do not cause acute bronchodilation in humans (Grootendorst et al.,
2003), any improvements in lung function would not likely result from enhanced ASM relaxation. While SABAs and LABAs do not independently inhibit inflammation when administered to patients with COPD or asthma, the enhancement of cAMP level in target cells may increase the anti-inflammatory effects of PDE4 and PDE3 inhibitors
(Giembycz and Newton, 2011). This was found in the studies reported here where formoterol-induced gene expression in BEAS-2B cells was sensitised by PDE3 plus
87 PDE4 inhibitors (Fig. 3.9 D-F) and in some cases gene expression was prolonged (Fig.
3.8 A and C). Likewise, it has been reported by others that CREB phosphorylation was significantly increased in fibroblasts from pde4d-deficient mice after the addition of β2- adrenoceptor agonist, when compared to fibroblasts from wild-type animals (Bruss et al., 2008). Similarly, our data demonstrated that combination of siguazodan plus rolipram significantly increased formoterol’s effect on CREB phosphorylation in BEAS-
2B cells (Fig. 3.7). Furthermore, the SABAs, salbutamol, and procaterol, act in combination with rolipram in monocytes to promote cAMP accumulation and PKA stimulation (Seldon et al., 1995). Moreover, our data show that cAMP-elevating agents
(formoterol, siguazodan and rolipram) induce CRE-dependent transcription via PKA activation (Fig. 3.5).
Roflumilast is the first PDE4 inhibitor to be marketed and is indicated for treating patients with severe, bronchitic COPD that suffer frequent acute exacerbations (Price et al., 2010). However, the use of roflumilast is still limited by its adverse side-effect profile
(Calverley et al., 2009; Fabbri et al., 2009). It is reasonable to speculate that the addition of a PDE3 inhibitor, which acts on the same cAMP/PKA/CREB signalling pathway (Fig. 3.5 B and 3.7 B), could be used to minimize the dose of a PDE4 inhibitor necessary to achieve a clinical benefit. Thus, inhibitors of PDE combination therapies could provide extra symptomatic benefit in certain patient populations, especially those for whom the symptoms are not completely controlled by LABA (Giembycz and Newton,
2011).
88 4.5 PDE inhibitors and glucocorticoids
While the transrepression of inflammatory transcription factors may play role in the anti- inflammatory effects of glucocorticoids, many studies also suggest an important role for gene transcription in the anti-inflammatory actions of glucocorticoids (Newton and
Holden, 2007). Accordingly, glucocorticoid-dependent stimulation of anti-inflammatory gene expression may represent a major mechanism of glucocorticoid-mediated inflammation suppression. The data presented in this study demonstrates that in BEAS-
2B cells, dexamethasone independently activated the expression of MKP-1, CRISPLD2,
CD200, and RGS2. Several studies reveal that β2-adrenoceptor agonists increase the action of glucocorticoids by means of cAMP/PKA signalling cascade (Ammit et al.,
2002; Hallsworth et al., 2001; Kaur et al., 2008). Based on these results, other cAMP- elevating drugs such as PDE3 and/or PDE4 inhibitors are likely to augment glucocorticoid action. As shown in Figure 3.12 A, B and C, our data demonstrate that siguazodan plus rolipram increased the expression of glucocorticoid-inducible genes.
However, these effects were small in comparison with other data showing that cAMP- elevating agents, such as LABA, and glucocorticoids fully enhanced the expression of
RGS2 and MKP-1 in BEAS-2B cells (Kaur et al., 2008). Our results were probably affected by low baseline levels of cAMP in BEAS-2B cells, thus necessitating the presence of a stimulus of cAMP formation for studying the interaction between PDE inhibitors and glucocorticoids on these genes. When we added sub-maximal concentrations of formoterol to the system, we witnessed a functional interaction between siguazodan, rolipram, formoterol and dexamethasone. Siguazodan plus rolipram sensitised the expression of LABA-induced and/or glucocorticoid-inducible
89 genes (Fig. 3.9 and 3.12). Moreover, siguazodan plus rolipram sustained the expression of some of these genes (RGS2 and CD200) for longer periods of time (Fig. 3.8 A and C and 3.10 A and C).
Mechanism of Interaction
According to our investigation with the PKIα expression vector, CRE-dependent transcription is induced by formoterol, siguazodan, and rolipram via stimulation of the canonical cAMP/PKA signalling pathway (Fig. 3.5). Moreover, other data demonstrated that the triggering of this pathway enabled PDE4 inhibitors to enhance GRE-dependent transcription. However, the precise molecular targets of PKA have not yet been identified (Moodley et al., 2013). Different possible mechanisms have been suggested for describing the ability of LABA to enhance the effect of glucocorticoid. For example, cAMP-elevating agents might enhance the translocation of GRs from the cytosol to the nucleus (Miller et al., 2002). While evidence for this idea is available, it would imply that cAMP-elevating agents should increase the transcription of all glucocorticoid-inducible genes, which is not the case (Kaur et al., 2008). For instance, the transcriptional regulator, glucocorticoid-inducible leucine zipper (GILZ), assists in suppressing inflammatory genes and is highly glucocorticoid-inducible in airway epithelial cells
(Eddleston et al., 2007). In Kaur et al. (2008), cAMP-elevating agents, including forskolin or salmeterol were unable to increases the ability of glucocorticoid to enhance the expression of GLIZ, although other anti-inflammatory genes such as kinase inhibitor protein 2 of 57kDa (p57KIP2) and MKP-1, were enhance maximally by the addition of cAMP-elevating agents (Kaur et al., 2008; Newton et al., 2010). Similarly, our data demonstrate that PDE inhibitors only succeeded in sustaining the expression of two
90 glucocorticoid-inducible genes, RGS2 and CD200. Since cAMP-elevating agents only enhanced the expression of a particular subset of glucocorticoid genes, other explanations must be considered for how they modulate GR signalling (Moodley et al.,
2013; Newton et al., 2010). First, several parts of the transcriptional machinery may be subject to PKA-mediated phosphorylation. Since the human GR has many putative PKA phosphorylation sites, phospho-GR may possess an enhanced capacity to stimulate the transcription of some, but not all, glucocorticoid-inducible genes (Galliher-Beckley and
Cidlowski, 2009; Miller et al., 2007). An alternative explanation suggests that PKA might phosphorylate substrates that exist downstream of the GR that, for example, control the activity of particular cofactors (Moyer et al., 1993). Although the regulation of glucocorticoid-inducible genes remains unclear, the processes of phosphorylation may be promoter specific and interpret why the expression of only some genes are positively and synergistically enhanced by cAMP-elevating agents (Moodley et al., 2013).
Other anti-inflammatory genes
While we only studied five genes, microarray analysis demonstrates that additional genes possessing the capacity for anti-inflammatory effects, such as p57KIP2, can be induced by glucocorticoid and cAMP-elevating agents (Kaur et al., 2008). p57KIP2 constitutes part of the second family of cyclin-dependent kinases (CDKs) (Lee et al.,
1995; Samuelsson et al., 1999). As a cycle-cell inhibitor, this protein attaches to and prevents CDK-cyclin complexes in all stages of cell cycling (Lee et al., 1995;
Samuelsson et al., 1999). Furthermore, p57KIP2 also binds to proliferating cell nuclear antigen, thereby suppressing DNA replication (Watanabe et al., 1998). Consequently, the expression of p57KIP2 in certain cells could inhibit mitogenesis and suppress airway
91 remodeling, which are common characteristics of COPD (Newton et al., 2010). We can hypothesize that the presence of PDE3 plus PDE4 inhibitors can stimulate further the expression of p57KIP2. Indeed, other studies have shown that in BEAS-2B cells, a LABA and a glucocorticoid induced p57KIP2, which was further augmented in the presence of a
PDE4 inhibitor (Moodley et al., 2013).
All experiments in this thesis involved the use of one pulmonary epithelial cell line.
Critics may argue that the sole induction of anti-inflammatory genes in epithelial cells indicates that such genes would fail to have an impact on the total anti-inflammatory action in COPD. However, studies have provided evidence of expression of anti- inflammatory genes in several other cells. In particular, MKP-1 undergoes expression in a significant variety of cell types, such as airway smooth muscle, macrophages, mast cells, and epithelial cells (Abraham et al., 2006; Clark et al., 2008; Kassel et al., 2001;
Kaur et al., 2008). Similarly, CD200 is expressed in dendritic cells, B cells, T-cells and primary airway epithelial cells (Holt and Strickland, 2008; Snelgrove et al., 2008) whereas RGS2 has been identified in B lymphocytes (Reif and Cyster, 2000) dendritic cells (Shi et al., 2004) primary airway epithelial cells (Moodley et al., 2013) and ASM
(Holden et al., 2011).
While the PDE inhibitors used in our study failed to induce, enhance or prolong the expression of MKP-1 in BEAS-2B cells, in another study it was reported that rolipram induced the expression of MKP-1 in macrophages (Korhonen et al., 2013). From this, it is reasonable to speculate that PDE inhibitors alone or in combination with β2- adrenoceptor agonists and glucocorticoids can enhance further the expression of MKP-
1 in other cell types involved in COPD.
92 An area that is not addressed in the current study is the possibility that cAMP-elevating agents may increase the capacity of ICSs to transactivate side-effect genes. In fact, both cAMP and glucocorticoids regulate several metabolic genes (Newton et al., 2010).
For instance, cAMP-elevating stimuli and glucocorticoids act synergistically to induce the expression of phosphoenol pyruvate carboxykinase (PEPCK), an enzyme responsible for controlling gluconeogenesis (Yeagley and Quinn, 2005). Moreover, glucocorticoids can independently induce MKP-1 expression and this expression is increased further with the presence of cAMP-elevating agents (Kassel et al., 2001; Kaur et al., 2008). While MKP-1 has very important role in suppression of inflammation, it is also can suppress osteoblast proliferation, leading to the development of osteoporosis
(Horsch et al., 2007). Thus, cAMP-elevating agents are expected to increase the osteoporotic actions of glucocorticoids (Newton et al., 2010). Alternatively, cAMP- elevating agents directly increase c-fos expression (Kellenberger et al., 1998). Thus, c- fos has the ability to induce the transcription of many biochemical marker of bone formation such as osteocalcin. This action subsequently represents the potential anabolic impact of such medications on osteoblasts, serving to protect bone against the osteoporotic actions of glucocorticoids (Kellenberger et al., 1998). For example, the
PDE4 inhibitor rolipram fulfills an anabolic function in bone formation in mice (Bonnet et al., 2007). Moreover, engagement of CD200/CD200R is reported to inhibit the production of pro-inflammatory cytokines and also stimulates TGF-β expression, which assists the bone formation by increasing the proliferation of osteoblast progenitors (Lee et al., 2006). Although the overall impact of combining cAMP-elevating agents and glucocorticoids on bone density remains unclear, these results suggest that the
93 undesirable induction of some genes by glucocorticoids may be counteracted by cAMP- elevating agents (Newton et al., 2010).
4.6 Clinical Relevance
This thesis contains a number of possibly significant implications for understanding and improving the treatment of COPD. Glucocorticoid monotherapies represent a relatively ineffective option for COPD patients, or patients with severe asthma who smoke cigarettes (Barnes and Adcock, 2009). Indeed, current research indicates that different aspects of GR signalling, such as the affinity of glucocorticoid for GR, as well as GR translocation and transcriptional activity, are suppressed by inflammatory stimuli (Irusen et al., 2002; Kam et al., 1993; Pariante et al., 1999). Specifically, stimuli such as cigarette smoke and TNFα reduce GRE dependent transcription, and TNFα substantially suppresses the glucocorticoid-dependent expression of p57KIP2 (Rider et al., 2011). Therefore, the down-regulation of anti-inflammatory genes that are induced by glucocorticoids may assist in explaining the association between glucocorticoid resistance and cigarette smoking or severe inflammation. Furthermore, LABAs are able to restore glucocorticoid-induced p57KIP2 mRNA expression to a level that was similar to that observed in the absence of TNFα (Rider et al., 2011). Moreover, the PDE4 inhibitor significantly enhanced further increased of p57KIP2 mRNA when added to airways cells treated with glucocorticoid and LABA compared to cells treated only with glucocorticoid and LABA (Moodley et al., 2013). These results imply that the addition of cAMP- elevating agents, including formoterol, siguazodan and rolipram can not only restore glucocorticoid-inducible genes to levels resembling those in the absence of inflammatory agents, but can also stimulate the expression of p57KIP2 mRNA to a
94 substantially higher level. Because the expression of this gene may contribute to the anti-inflammatory effects of glucocorticoids, its increased expression with LABA and
PDE3 and PDE4 inhibitors may impart further anti-inflammatory benefit. Furthermore, glucocorticoids have the ability to induce the expression of many genes including genes with anti-inflammatory effect. At the same time, cigarette smoke can inhibit many of those genes in a similar way to p57KIP2. Thus, it is reasonable to speculate that cAMP- elevating agents will have the ability also to restore the expression of numerous glucocorticoid-inducible genes.
Even when given the recommended maximum dose of a β2-adrenoceptor agonists and
ICS, possibly combined with a long-acting anticholinergic, many COPD patients still suffer exacerbations (Calverley et al., 2012). However, roflumilast, a PDE4 inhibitor, has recently been approved as an add-on therapy in patients with severe, bronchitic COPD who experience frequent exacerbations and who will generally be taking ICS/LABA combination therapies. Several studies have shown that roflumilast exhibited sustained clinical efficacy by enhancing lung function and decreasing severe exacerbation
(Calverley et al., 2012; Calverley et al., 2009). Accordingly, the current study provides a mechanistic basis for these therapeutic effects.
PDE3 plus PDE4 inhibitors together were able to induce expression of CD200 and
CRISPLD2 mRNA in BEAS-2B cells (Fig. 3.9 A-C). Moreover, the expression of CD200,
CRISPLD2 and RGS2 induced by glucocorticoid with a lower concentration of β2- adrenoceptor agonists were enhanced further upon the addition of inhibitors of PDE3 plus PDE4 (Fig. 3.12 D-F). Functionally, CD200 may fulfill an essential function in decreasing COPD symptoms. In COPD patients viral infections and bacteria can induce
95 prolonged excessive inflammatory reactions causing exacerbations, which may be driven by pulmonary macrophages. Expression of CD200 on epithelial or other airway cells can bind to CD200R on macrophages, resulting in the inhibition of inflammatory signalling (Snelgrove et al., 2008). This action may reduce the inflammation and exacerbations frequency in COPD. Moreover, infection of COPD patients with gram negative bacteria can promote an acute exacerbation of COPD; CRISPLD2 is a novel
LPS-binding protein whose up-regulation would be expected bind the LPS on the gram negative bacteria and prevents the activation of TLR4, which mediates pro-inflammatory responses (Wang et al., 2009).
The research presented in this thesis also found that PDE inhibitors reduced the rapid decline in RGS2 mRNA that occurs following the initial peak in expression at 1 h (Fig.
3.11). RGS2, a GAP, stimulates the hydrolysis of GTP to GDP thereby terminating Gq signalling (Heximer, 2004). Activation of Gq signalling by inflammatory mediator such as histamine and leukotrienes can increase the release of Ca2+ from intracellular stores causing ASM contraction (Heximer, 2004). Therefore, inhibition of Gq signalling by up- regulated RGS2 could protect the airways against constrictor stimuli and thereby improve lung function as is seen in COPD patients.
Moreover, histamine increases TNFα-stimulated IL-6 and IL-8 release and this effect of histamine occurs via H1 receptor which linked to Gq (Holden et al., 2007; Li et al., 2001).
RGS2 may combat these actions by inhibiting histamine H1 receptor Gq signalling. Thus, we suggest that RGS2 may fulfill a function in resolving inflammatory responses associated with COPD which could reduce exacerbation rates. Therefore, combination therapies that include an ICS, a LABA plus both PDE3 and PDE4 inhibitor might provide
96 superior effectiveness in the regulation of symptoms in diseases such as COPD and asthma. One thing to note is that the induction of CRISPLD2, CD200 and RGS2 were induced by PDE3 plus PDE4 inhibitors in combination, while the clinical data represent the effect of the PDE4 inhibitor, roflumilast, alone.
97 Chapter five: Future work
This thesis provides information that emphasizes some areas for further study:
First of all, the data presented in this study show that inhibitors of PDE4 and PDE3 were able to prolong and/or sensitise BEAS-2B cells to the expression of LABA/glucocorticoid inducible genes. We need to identify the exact role of PDE3 and PDE4 inhibitors alone relative to the combination of PDE 3 plus PDE4 inhibitors on the induction of those genes.
Second, this study utilized BEAS-2B cell lines to represent pulmonary bronchial epithelial cells. Although this cell line is well characterized model of bronchial epithelial cells, the responses measured may be different from those of primary epithelial cells.
Accordingly, the use of the BEAS-2B cell line indicates a possible limitation of this thesis. Future studies should verify the results of this thesis in primary epithelial cells and other relative cells such as ASM and/or T-lymphocytes cells.
Third, the sole use of dexamethasone as a glucocorticoid, and rolipram as a PDE4 inhibitor, do not correspond to the drugs used to treat COPD. Consequently, further investigations can be conducted with more clinically-relevant glucocorticoids and PDE4 inhibitor, such as budesonide or fluticasone propionate, and roflumilast respectively to confirm the relevance of the findings.
Additionally, further studies should examine the functional consequences of genes, such as CD200, RGS2, and CRISPLD2. In particular, studies investigating over- expression and small interfering RNA (siRNA) could evaluate changes in inflammatory responses that are associated with the specific genes. The functional consequences of
98 lacking such genes and alterations in the COPD could also undergo study by using knockout mice models.
Last, future investigations could assess biopsy samples from COPD patients receiving therapy from clinically-relevant combinations of medications to determine the expression of RGS2, CD200, and CRISPLD2.
99 References
Aaron, S.D., Vandemheen, K.L., Hebert, P., Dales, R., Stiell, I.G., Ahuja, J., Dickinson, G., Brison, R., Rowe, B.H., Dreyer, J., et al. (2003). Outpatient Oral Prednisone after Emergency Treatment of Chronic Obstructive Pulmonary Disease. New England Journal of Medicine 348, 2618-2625. Abbinante-Nissen, J.M., Simpson, L.G., and Leikauf, G.D. (1995). Corticosteroids increase secretory leukocyte protease inhibitor transcript levels in airway epithelial cells. Am J Physiol 268, L601-606. Abraham, S.M., and Clark, A.R. (2006). Dual-specificity phosphatase 1: a critical regulator of innate immune responses. Biochem Soc Trans 34, 1018-1023. Abraham, S.M., Lawrence, T., Kleiman, A., Warden, P., Medghalchi, M., Tuckermann, J., Saklatvala, J., and Clark, A.R. (2006). Antiinflammatory effects of dexamethasone are partly dependent on induction of dual specificity phosphatase 1. J Exp Med 203, 1883-1889. Adcock, I.M., Gilbey, T., Gelder, C.M., Chung, K.F., and Barnes, P.J. (1996). Glucocorticoid receptor localization in normal and asthmatic lung. Am J Respir Crit Care Med 154, 771-782. Akerblom, I.E., Slater, E.P., Beato, M., Baxter, J.D., and Mellon, P.L. (1988). Negative regulation by glucocorticoids through interference with a cAMP responsive enhancer. Science 241, 350-353. Ammit, A.J., Lazaar, A.L., Irani, C., O'Neill, G.M., Gordon, N.D., Amrani, Y., Penn, R.B., and Panettieri, R.A., Jr. (2002). Tumor necrosis factor-alpha-induced secretion of RANTES and interleukin-6 from human airway smooth muscle cells: modulation by glucocorticoids and beta-agonists. Am J Respir Cell Mol Biol 26, 465-474. Anderson, G.P. (1993). Long acting inhaled beta-adrenoceptor agonists the comparative pharmacology of formoterol and salmeterol. Agents Actions Suppl 43, 253-269. Anderson, G.P. (1995). Adrenaline and noradrenaline. In: Raeburn D, Giembycz MA, eds. Airways Smooth Muscle: Neurotransmitters, Amines, Lipid Mediators and Signal Transduction. Basle, Birkhauser Verlag, 1995; pp. 1–79 Andrisani, O.M. (1999). CREB-mediated transcriptional control. Crit Rev Eukaryot Gene Expr 9, 19-32. Banner, K.H., and Press, N.J. (2009). Dual PDE3/4 inhibitors as therapeutic agents for chronic obstructive pulmonary disease. Br J Pharmacol 157, 892-906. Bardin, P.G., Dorward, M.A., Lampe, F.C., Franke, B., and Holgate, S.T. (1998). Effect of selective phosphodiesterase 3 inhibition on the early and late asthmatic responses to inhaled allergen. Br J Clin Pharmacol 45, 387-391. Barisione, G., Baroffio, M., Crimi, E., and Brusasco, V. (2010). Beta-Adrenergic Agonists. Pharmaceuticals 3, 1016-1044. Barnes, N.C., Qiu, Y.-S., Pavord, I.D., Parker, D., Davis, P.A., Zhu, J., Johnson, M., Thomson, N.C., Jeffery, P.K., and Group, o.b.o.t.S.S. (2006). Antiinflammatory Effects of Salmeterol/Fluticasone Propionate in Chronic Obstructive Lung Disease. American Journal of Respiratory and Critical Care Medicine 173, 736-743.
100 Barnes, P.J. (2002). Scientific rationale for inhaled combination therapy with long-acting beta2-agonists and corticosteroids. Eur Respir J 19, 182-191. Barnes, P.J. (2003). Therapy of chronic obstructive pulmonary disease. Pharmacology & Therapeutics 97, 87-94. Barnes, P.J. (2004a). Alveolar Macrophages as Orchestrators of COPD. COPD: Journal of Chronic Obstructive Pulmonary Disease 1, 59-70. Barnes, P.J. (2004b). Mediators of Chronic Obstructive Pulmonary Disease. Pharmacological Reviews 56, 515-548. Barnes, P.J. (2004c). The role of anticholinergics in chronic obstructive pulmonary disease. Am J Med 20, 24S-32S. Barnes, P.J. (2006a). Corticosteroid effects on cell signalling. Eur Respir J 27, 413-426. Barnes, P.J. (2006b). How corticosteroids control inflammation: Quintiles Prize Lecture 2005. British Journal of Pharmacology 148, 245-254. Barnes, P.J. (2008). Immunology of asthma and chronic obstructive pulmonary disease. Nat Rev Immunol 8, 183-192. Barnes, P.J. (2010). Mechanisms and resistance in glucocorticoid control of inflammation. J Steroid Biochem Mol Biol 120, 76-85. Barnes, P.J. (2011). Glucocorticosteroids: current and future directions. Br J Pharmacol 163, 29-43. Barnes, P.J., and Adcock, I.M. (2009). Glucocorticoid resistance in inflammatory diseases. Lancet 373, 1905-1917. Baur, F., Beattie, D., Beer, D., Bentley, D., Bradley, M., Bruce, I., Charlton, S.J., Cuenoud, B., Ernst, R., Fairhurst, R.A., et al. (2010). The identification of indacaterol as an ultralong-acting inhaled beta2-adrenoceptor agonist. J Med Chem 53, 3675-3684. Bender, A.T., and Beavo, J.A. (2006). Cyclic nucleotide phosphodiesterases: molecular regulation to clinical use. Pharmacol Rev 58, 488-520. Bergmann, M.W., Staples, K.J., Smith, S.J., Barnes, P.J., and Newton, R. (2004). Glucocorticoid inhibition of granulocyte macrophage-colony-stimulating factor from T cells is independent of control by nuclear factor-kappaB and conserved lymphokine element 0. Am J Respir Cell Mol Biol 30, 555-563. Billington, C., and Penn, R. (2003). Signaling and regulation of G protein-coupled receptors in airway smooth muscle. Respiratory Research 4, 2. Billington, C.K., Hall, I.P., Mundell, S.J., Parent, J.L., Panettieri, R.A., Jr., Benovic, J.L., and Penn, R.B. (1999). Inflammatory and contractile agents sensitize specific adenylyl cyclase isoforms in human airway smooth muscle. Am J Respir Cell Mol Biol 21, 597-606. Bonnet, N., Bernard, P., Beaupied, H., Bizot, J.C., Trovero, F., Courteix, D., and Benhamou, C.L. (2007). Various effects of antidepressant drugs on bone microarchitectecture, mechanical properties and bone remodeling. Toxicol Appl Pharmacol 221, 111-118. Bos, I.S., Gosens, R., Zuidhof, A.B., Schaafsma, D., Halayko, A.J., Meurs, H., and Zaagsma, J. (2007). Inhibition of allergen-induced airway remodelling by tiotropium and budesonide: a comparison. Eur Respir J 30, 653-661.
101 Bos, J.L. (2003). Epac: a new cAMP target and new avenues in cAMP research. Nat Rev Mol Cell Biol 4, 733-738. Boswell-Smith, V., Cazzola, M., and Page, C.P. (2006). Are phosphodiesterase 4 inhibitors just more theophylline? J Allergy Clin Immunol 117, 1237-1243. Bourbeau, J., Christodoulopoulos, P., Maltais, F., Yamauchi, Y., Olivenstein, R., and Hamid, Q. (2007). Effect of salmeterol/fluticasone propionate on airway inflammation in COPD: a randomised controlled trial. Thorax 62, 938-943. Boutros, T., Chevet, E., and Metrakos, P. (2008). Mitogen-activated protein (MAP) kinase/MAP kinase phosphatase regulation: roles in cell growth, death, and cancer. Pharmacol Rev 60, 261-310. Brittain, R.T. (1990). Approaches to a long-acting, selective beta 2-adrenoceptor stimulant. Lung 168, 111-114. Brown, W.M. (2007). Treating COPD with PDE 4 inhibitors. Int J Chron Obstruct Pulmon Dis 2, 517-533. Bruss, M.D., Richter, W., Horner, K., Jin, S.L., and Conti, M. (2008). Critical role of PDE4D in beta2-adrenoceptor-dependent cAMP signaling in mouse embryonic fibroblasts. J Biol Chem 283, 22430-22442. Buhl, R., and Farmer, S.G. (2005). Future Directions in the Pharmacologic Therapy of Chronic Obstructive Pulmonary Disease. Proceedings of the American Thoracic Society 2, 83-93. Buhling, F., Lieder, N., Kuhlmann, U.C., Waldburg, N., and Welte, T. (2007). Tiotropium suppresses acetylcholine-induced release of chemotactic mediators in vitro. Respir Med 101, 2386-2394. Bundschuh, D.S., Eltze, M., Barsig, J., Wollin, L., Hatzelmann, A., and Beume, R. (2001). In Vivo Efficacy in Airway Disease Models of Roflumilast, a Novel Orally Active PDE4 Inhibitor. Journal of Pharmacology and Experimental Therapeutics 297, 280-290. Burge, P.S., Calverley, P.M., Jones, P.W., Spencer, S., Anderson, J.A., and Maslen, T.K. (2000). Randomised, double blind, placebo controlled study of fluticasone propionate in patients with moderate to severe chronic obstructive pulmonary disease: the ISOLDE trial. BMJ 320, 1297-1303. Calverley, P.M., Martinez, F.J., Fabbri, L.M., Goehring, U.M., and Rabe, K.F. (2012). Does roflumilast decrease exacerbations in severe COPD patients not controlled by inhaled combination therapy? The REACT study protocol. Int J Chron Obstruct Pulmon Dis 7, 375-382. Calverley, P.M., Rabe, K.F., Goehring, U.M., Kristiansen, S., Fabbri, L.M., and Martinez, F.J. (2009). Roflumilast in symptomatic chronic obstructive pulmonary disease: two randomised clinical trials. Lancet 374, 685-694. Calverley, P.M.A., Anderson, J.A., Celli, B., Ferguson, G.T., Jenkins, C., Jones, P.W., Yates, J.C., and Vestbo, J.r. (2007). Salmeterol and Fluticasone Propionate and Survival in Chronic Obstructive Pulmonary Disease. New England Journal of Medicine 356, 775-789. Carstairs, J.R., Nimmo, A.J., and Barnes, P.J. (1984). Autoradiographic localisation of beta-adrenoceptors in human lung. Eur J Pharmacol 103, 189-190.
102 Cazzola, M., Di Perna, F., D'Amato, M., Califano, C., Matera, M.G., and D'Amato, G. (2001). Formoterol Turbuhaler for as-needed therapy in patients with mild acute exacerbations of COPD. Respir Med 95, 917-921. Celli, B.R., and MacNee, W. (2004). Standards for the diagnosis and treatment of patients with COPD: a summary of the ATS/ERS position paper. Eur Respir J 23, 932-946. Chanez, P., Vignola, A.M., O'Shaugnessy, T., Enander, I., Li, D., Jeffery, P.K., and Bousquet, J. (1997). Corticosteroid reversibility in COPD is related to features of asthma. American Journal of Respiratory and Critical Care Medicine 155, 1529-1534. Chivers, J.E., Cambridge, L.M., Catley, M.C., Mak, J.C., Donnelly, L.E., Barnes, P.J., and Newton, R. (2004). Differential effects of RU486 reveal distinct mechanisms for glucocorticoid repression of prostaglandin E2 release. European Journal of Biochemistry 271, 4042-4052. Clark, A.R. (2003). MAP kinase phosphatase 1: a novel mediator of biological effects of glucocorticoids? J Endocrinol 178, 5-12. Clark, A.R. (2007). Anti-inflammatory functions of glucocorticoid-induced genes. Molecular and Cellular Endocrinology 275, 79-97. Clark, A.R., and Belvisi, M.G. (2012). Maps and legends: the quest for dissociated ligands of the glucocorticoid receptor. Pharmacol Ther 134, 54-67. Clark, A.R., Martins, J.R., and Tchen, C.R. (2008). Role of dual specificity phosphatases in biological responses to glucocorticoids. J Biol Chem 283, 25765-25769. Clark, A.R., Martins, J.R., and Tchen, C.R. (2008). Role of dual specificity phosphatases in biological responses to glucocorticoids. J Biol Chem 283, 25765-25769. Conti, M., and Beavo, J. (2007). Biochemistry and physiology of cyclic nucleotide phosphodiesterases: essential components in cyclic nucleotide signaling. Annu Rev Biochem 76, 481-511. Conti, M.A., and Adelstein, R.S. (1981). The relationship between calmodulin binding and phosphorylation of smooth muscle myosin kinase by the catalytic subunit of 3':5' cAMP-dependent protein kinase. J Biol Chem 256, 3178-3181. Culpitt, S.V., Rogers, D.F., Shah, P., De Matos, C., Russell, R.E.K., Donnelly, L.E., and Barnes, P.J. (2003). Impaired Inhibition by Dexamethasone of Cytokine Release by Alveolar Macrophages from Patients with Chronic Obstructive Pulmonary Disease. American Journal of Respiratory and Critical Care Medicine 167, 24-31. Davies, L., Angus, R.M., and Calverley, P.M.A. (1999). Oral corticosteroids in patients admitted to hospital with exacerbations of chronic obstructive pulmonary disease: a prospective randomised controlled trial. The Lancet 354, 456-460. de Lanerolle, P., Nishikawa, M., Yost, D.A., and Adelstein, R.S. (1984). Increased phosphorylation of myosin light chain kinase after an increase in cyclic AMP in intact smooth muscle. Science 223, 1415-1417. de Rooij, J., Zwartkruis, F.J., Verheijen, M.H., Cool, R.H., Nijman, S.M., Wittinghofer, A., and Bos, J.L. (1998). Epac is a Rap1 guanine-nucleotide- exchange factor directly activated by cyclic AMP. Nature 396, 474-477.
103 Denis, D., and Riendeau, D. (1999). Phosphodiesterase 4-dependent regulation of cyclic AMP levels and leukotriene B4 biosynthesis in human polymorphonuclear leukocytes. Eur J Pharmacol 367, 343-350. Dent, G., White, S.R., Tenor, H., Bodtke, K., Schudt, C., Leff, A.R., Magnussen, H., and Rabe, K.F. (1998). Cyclic nucleotide phosphodiesterase in human bronchial epithelial cells: characterization of isoenzymes and functional effects of PDE inhibitors. Pulm Pharmacol Ther 11, 47-56. Deroo, B.J., and Archer, T.K. (2001). Glucocorticoid receptor-mediated chromatin remodeling in vivo. Oncogene 20, 3039-3046. Dhami, R., Gilks, B., Xie, C., Zay, K., Wright, J.L., and Churg, A. (2000). Acute Cigarette Smoke–Induced Connective Tissue Breakdown Is Mediated by Neutrophils and Prevented by α 1-Antitrypsin. American Journal of Respiratory Cell and Molecular Biology 22, 244-252. Di Stefano, A., Caramori, G., Capelli, A., Gnemmi, I., Ricciardolo, F.L., Oates, T., Donner, C.F., Chung, K.F., Barnes, P.J., and Adcock, I.M. (2004). STAT4 activation in smokers and patients with chronic obstructive pulmonary disease. European Respiratory Journal 24, 78-85. Doll, R., Peto, R., Boreham, J., and Sutherland, I. (2004). Mortality in relation to smoking: 50 years' observations on male British doctors. BMJ 328, 1519. Eddleston, J., Herschbach, J., Wagelie-Steffen, A.L., Christiansen, S.C., and Zuraw, B.L. (2007). The anti-inflammatory effect of glucocorticoids is mediated by glucocorticoid-induced leucine zipper in epithelial cells. J Allergy Clin Immunol 119, 115-122. Eickelberg, O., Roth, M., Lorx, R., Bruce, V., Rudiger, J., Johnson, M., and Block, L.H. (1999). Ligand-independent activation of the glucocorticoid receptor by beta2-adrenergic receptor agonists in primary human lung fibroblasts and vascular smooth muscle cells. J Biol Chem 274, 1005-1010. Fabbri, L.M., Calverley, P.M., Izquierdo-Alonso, J.L., Bundschuh, D.S., Brose, M., Martinez, F.J., and Rabe, K.F. (2009). Roflumilast in moderate-to-severe chronic obstructive pulmonary disease treated with longacting bronchodilators: two randomised clinical trials. Lancet 374, 695-703. Fan Chung, K. (2006). Phosphodiesterase inhibitors in airways disease. Eur J Pharmacol 533, 110-117. Flower, R.J., and Rothwell, N.J. (1994). Lipocortin-1: cellular mechanisms and clinical relevance. Trends Pharmacol Sci 15, 71-76. Fuhrmann, M., Jahn, H.U., Seybold, J., Neurohr, C., Barnes, P.J., Hippenstiel, S., Kraemer, H.J., and Suttorp, N. (1999). Identification and function of cyclic nucleotide phosphodiesterase isoenzymes in airway epithelial cells. Am J Respir Cell Mol Biol 20, 292-302. Fujimura, M., Kamio, Y., Saito, M., Hashimoto, T., and Matsuda, T. (1995). Bronchodilator and bronchoprotective effects of cilostazol in humans in vivo. Am J Respir Crit Care Med 151, 222-225. Galliher-Beckley, A.J., and Cidlowski, J.A. (2009). Emerging roles of glucocorticoid receptor phosphorylation in modulating glucocorticoid hormone action in health and disease. IUBMB Life 61, 979-986.
104 Gantner, F., Schudt, C., Wendel, A., and Hatzelmann, A. (1999). Characterization of the phosphodiesterase (PDE) pattern of in vitro-generated human dendritic cells (DC) and the influence of PDE inhibitors on DC function. Pulm Pharmacol Ther 12, 377-386. Gao, H., Hoesel, L.M., Guo, R.-F., Rancilio, N.J., Sarma, J.V., and Ward, P.A. (2006). Adenoviral-Mediated Overexpression of SOCS3 Enhances IgG Immune Complex-Induced Acute Lung Injury. The Journal of Immunology 177, 612-620. Giembycz, M.A. (1996). Phosphodiesterase 4 and tolerance to beta 2- adrenoceptor agonists in asthma. Trends Pharmacol Sci 17, 331-336. Giembycz, M.A., and Newton, R. (2006). Beyond the dogma: novel beta2- adrenoceptor signalling in the airways. Eur Respir J 27, 1286-1306. Giembycz, M.A., and Newton, R. (2011). Harnessing the clinical efficacy of phosphodiesterase 4 inhibitors in inflammatory lung diseases: dual-selective phosphodiesterase inhibitors and novel combination therapies. Handb Exp Pharmacol 2011, 415-446. Giembycz, M.A., Corrigan, C.J., Seybold, J., Newton, R., and Barnes, P.J. (1996). Identification of cyclic AMP phosphodiesterases 3, 4 and 7 in human CD4+ and CD8+ T-lymphocytes: role in regulating proliferation and the biosynthesis of interleukin-2. Br J Pharmacol 118, 1945-1958. Giguere, V., Hollenberg, S.M., Rosenfeld, M.G., and Evans, R.M. (1986). Functional domains of the human glucocorticoid receptor. Cell 46, 645-652. Gonzalez, G.A., and Montminy, M.R. (1989). Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133. Cell 59, 675-680. Grootendorst, D.C., Gauw, S.A., Baan, R., Kelly, J., Murdoch, R.D., Sterk, P.J., and Rabe, K.F. (2003). Does a single dose of the phosphodiesterase 4 inhibitor, cilomilast (15 mg), induce bronchodilation in patients with chronic obstructive pulmonary disease? Pulm Pharmacol Ther 16, 115-120. Grootendorst, D.C., Gauw, S.A., Verhoosel, R.M., Sterk, P.J., Hospers, J.J., Bredenbröker, D., Bethke, T.D., Hiemstra, P.S., and Rabe, K.F. (2007). Reduction in sputum neutrophil and eosinophil numbers by the PDE4 inhibitor roflumilast in patients with COPD. Thorax 62, 1081-1087. Gross, N.J., and Skorodin, M.S. (1984). Role of the parasympathetic system in airway obstruction due to emphysema. N Engl J Med 311, 421-425. Grumelli, S., Corry, D.B., Song, L.Z., Song, L., Green, L., Huh, J., Hacken, J., Espada, R., Bag, R., Lewis, D.E., and Kheradmand, F. (2004). An immune basis for lung parenchymal destruction in chronic obstructive pulmonary disease and emphysema. PLoS Med 1, 19. Hadcock, J.R., Wang, H.Y., and Malbon, C.C. (1989). Agonist-induced destabilization of beta-adrenergic receptor mRNA. Attenuation of glucocorticoid- induced up-regulation of beta-adrenergic receptors. J Biol Chem 264, 19928- 19933. Hallsworth, M.P., Twort, C.H., Lee, T.H., and Hirst, S.J. (2001). beta(2)- adrenoceptor agonists inhibit release of eosinophil-activating cytokines from human airway smooth muscle cells. Br J Pharmacol 132, 729-741.
105 Hamad, A.M., Clayton, A., Islam, B., and Knox, A.J. (2003). Guanylyl cyclases, nitric oxide, natriuretic peptides, and airway smooth muscle function. American Journal of Physiology - Lung Cellular and Molecular Physiology 285, L973-L983. Hanania, N.A., and Donohue, J.F. (2007). Pharmacologic Interventions in Chronic Obstructive Pulmonary Disease: Bronchodilators. Proceedings of the American Thoracic Society 4, 526-534. Hatzelmann, A., and Schudt, C. (2001). Anti-Inflammatory and Immunomodulatory Potential of the Novel PDE4 Inhibitor Roflumilast in Vitro. Journal of Pharmacology and Experimental Therapeutics 297, 267-279. Heximer, S.P. (2004). RGS2-mediated regulation of Gqalpha. Methods Enzymol 390, 65-82. Himmler, A., Stratowa, C., and Czernilofsky, A.P. (1993). Functional testing of human dopamine D1 and D5 receptors expressed in stable cAMP-responsive luciferase reporter cell lines. J Recept Res 13, 79-94. Holden, N.S., Bell, M.J., Rider, C.F., King, E.M., Gaunt, D.D., Leigh, R., Johnson, M., Siderovski, D.P., Heximer, S.P., Giembycz, M.A., and Newton, R. (2011). beta2-Adrenoceptor agonist-induced RGS2 expression is a genomic mechanism of bronchoprotection that is enhanced by glucocorticoids. Proc Natl Acad Sci U S A 108, 19713-19718. Holden, N.S., Gong, W., King, E.M., Kaur, M., Giembycz, M.A., and Newton, R. (2007). Potentiation of NF-kappaB-dependent transcription and inflammatory mediator release by histamine in human airway epithelial cells. Br J Pharmacol 152, 891-902. Holt, P.G., and Strickland, D.H. (2008). The CD200-CD200R axis in local control of lung inflammation (Nat Immunol. 2008 Sep;9(9):1011-3. doi: 10.1038/ni0908- 1011.). Horsch, K., de Wet, H., Schuurmans, M.M., Allie-Reid, F., Cato, A.C., Cunningham, J., Burrin, J.M., Hough, F.S., and Hulley, P.A. (2007). Mitogen- activated protein kinase phosphatase 1/dual specificity phosphatase 1 mediates glucocorticoid inhibition of osteoblast proliferation. Mol Endocrinol 21, 2929-2940. Houslay, M.D. (2010). Underpinning compartmentalised cAMP signalling through targeted cAMP breakdown. Trends Biochem Sci 35, 91-100. Huang, L.Y., Tholanikunnel, B.G., Vakalopoulou, E., and Malbon, C.C. (1993). The M(r) 35,000 beta-adrenergic receptor mRNA-binding protein induced by agonists requires both an AUUUA pentamer and U-rich domains for RNA recognition. J Biol Chem 268, 25769-25775. Irusen, E., Matthews, J.G., Takahashi, A., Barnes, P.J., Chung, K.F., and Adcock, I.M. (2002). p38 Mitogen-activated protein kinase-induced glucocorticoid receptor phosphorylation reduces its activity: role in steroid-insensitive asthma. J Allergy Clin Immunol 109, 649-657. Issa, R., Xie, S., Khorasani, N., Sukkar, M., Adcock, I.M., Lee, K.-Y., and Chung, K.F. (2007). Corticosteroid Inhibition of Growth-Related Oncogene Protein-α via Mitogen-Activated Kinase Phosphatase-1 in Airway Smooth Muscle Cells. The Journal of Immunology 178, 7366-7375. Ito, K., Lim, S., Caramori, G., Cosio, B., Chung, K.F., Adcock, I.M., and Barnes, P.J. (2002). A molecular mechanism of action of theophylline: Induction of
106 histone deacetylase activity to decrease inflammatory gene expression. Proceedings of the National Academy of Sciences 99, 8921-8926. Jeffery, P.K. (1998). Structural and inflammatory changes in COPD: a comparison with asthma. Thorax 53, 129-136. Jenkins, B.D., Pullen, C.B., and Darimont, B.D. (2001). Novel glucocorticoid receptor coactivator effector mechanisms. Trends Endocrinol Metab 12, 122-126. Jenkins, C., Jones, P., Calverley, P., Celli, B., Anderson, J., Ferguson, G., Yates, J., Willits, L., and Vestbo, J. (2009). Efficacy of salmeterol/fluticasone propionate by GOLD stage of chronic obstructive pulmonary disease: analysis from the randomised, placebo-controlled TORCH study. Respiratory Research 10, 59. Jin, S.L., and Conti, M. (2002). Induction of the cyclic nucleotide phosphodiesterase PDE4B is essential for LPS-activated TNF-alpha responses. Proc Natl Acad Sci U S A 99, 7628-7633. Johnson, G.L., and Lapadat, R. (2002). Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science 298, 1911- 1912. Johnson, M. (2001). Beta2-adrenoceptors: mechanisms of action of beta2- agonists. Paediatr Respir Rev 2, 57-62. Johnson, M. (2002). Effects of beta2-agonists on resident and infiltrating inflammatory cells. J Allergy Clin Immunol 110, S282-290. Johnson, M. (2004). Interactions between corticosteroids and beta2-agonists in asthma and chronic obstructive pulmonary disease. Proc Am Thorac Soc 1, 200- 206. Johnson, M., Butchers, P.R., Coleman, R.A., Nials, A.T., Strong, P., Sumner, M.J., Vardey, C.J., and Whelan, C.J. (1993). The pharmacology of salmeterol. Life Sci 52, 2131-2143. Kam, J.C., Szefler, S.J., Surs, W., Sher, E.R., and Leung, D.Y. (1993). Combination IL-2 and IL-4 reduces glucocorticoid receptor-binding affinity and T cell response to glucocorticoids. J Immunol 151, 3460-3466. Kankaanranta, H., Lindsay, M.A., Giembycz, M.A., Zhang, X., Moilanen, E., and Barnes, P.J. (2000). Delayed eosinophil apoptosis in asthma. J Allergy Clin Immunol 106, 77-83. Kardos, P., Wencker, M., Glaab, T., and Vogelmeier, C. (2007). Impact of Salmeterol/Fluticasone Propionate versus Salmeterol on Exacerbations in Severe Chronic Obstructive Pulmonary Disease. American Journal of Respiratory and Critical Care Medicine 175, 144-149. Kassel, O., Sancono, A., Kratzschmar, J., Kreft, B., Stassen, M., and Cato, A.C. (2001). Glucocorticoids inhibit MAP kinase via increased expression and decreased degradation of MKP-1. Embo J 20, 7108-7116. Katsunuma, T., Mak, J.C., and Barnes, P.J. (1998). Glucocorticoids reduce tachykinin NK2 receptor expression in bovine tracheal smooth muscle. Eur J Pharmacol 344, 99-106. Katsunuma, T., Roffel, A.F., Elzinga, C.R., Zaagsma, J., Barnes, P.J., and Mak, J.C. (1999). beta(2)-adrenoceptor agonist-induced upregulation of tachykinin NK(2) receptor expression and function in airway smooth muscle. Am J Respir Cell Mol Biol 21, 409-417.
107 Kaur, M., Chivers, J.E., Giembycz, M.A., and Newton, R. (2008). Long-acting beta2-adrenoceptor agonists synergistically enhance glucocorticoid-dependent transcription in human airway epithelial and smooth muscle cells. Mol Pharmacol 73, 203-214. Kawasaki, H., Springett, G.M., Mochizuki, N., Toki, S., Nakaya, M., Matsuda, M., Housman, D.E., and Graybiel, A.M. (1998). A family of cAMP-binding proteins that directly activate Rap1. Science 282, 2275-2279. Kellenberger, S., Muller, K., Richener, H., and Bilbe, G. (1998). Formoterol and isoproterenol induce c-fos gene expression in osteoblast-like cells by activating beta2-adrenergic receptors. Bone 22, 471-478. Kelsen, S.G., Higgins, N.C., Zhou, S., Mardini, I.A., and Benovic, J.L. (1995). Expression and function of the beta-adrenergic receptor coupled-adenylyl cyclase system on human airway epithelial cells. Am J Respir Crit Care Med 152, 1774-1783. Keravis, T., and Lugnier, C. (2011). Cyclic nucleotide phosphodiesterase (PDE) isozymes as targets of the intracellular signalling network: benefits of PDE inhibitors in various diseases and perspectives for future therapeutic developments. Br J Pharmacol 165, 1288-1305. Kimple, A.J., Soundararajan, M., Hutsell, S.Q., Roos, A.K., Urban, D.J., Setola, V., Temple, B.R.S., Roth, B.L., Knapp, S., Willard, F.S., and Siderovski, D.P. (2009). Structural Determinants of G-protein alpha Subunit Selectivity by Regulator of G-protein Signaling 2 (RGS2). Journal of Biological Chemistry 284, 19402-19411. King, E.M., Chivers, J.E., Rider, C.F., Minnich, A., Giembycz, M.A., and Newton, R. (2013). Glucocorticoid Repression of Inflammatory Gene Expression Shows Differential Responsiveness by Transactivation- and Transrepression-Dependent Mechanisms. PLoS ONE 8, e53936. Korhonen, R., Hömmö, T., Keränen, T., Laavola, M., Hämäläinen, M., Vuolteenaho, K., Lehtimäki, L., Kankaanranta, H., and Moilanen, E. (2013). Attenuation of TNF production and experimentally-induced inflammation by phosphodiesterase 4 inhibitor rolipram is mediated by mitogen-activated protein kinase phosphatase-1. British Journal of Pharmacology. Korn, S.H., Jerre, A., and Brattsand, R. (2001). Effects of formoterol and budesonide on GM-CSF and IL-8 secretion by triggered human bronchial epithelial cells. Eur Respir J 17, 1070-1077. Koto, H., Mak, J.C., Haddad, E.B., Xu, W.B., Salmon, M., Barnes, P.J., and Chung, K.F. (1996). Mechanisms of impaired beta-adrenoceptor-induced airway relaxation by interleukin-1beta in vivo in the rat. J Clin Invest 98, 1780-1787. Kovala, T., Sanwal, B.D., and Ball, E.H. (1997). Recombinant expression of a type IV, cAMP-specific phosphodiesterase: characterization and structure- function studies of deletion mutants. Biochemistry 36, 2968-2976. Koyama, S., Rennard, S.I., and Robbins, R.A. (1992). Acetylcholine stimulates bronchial epithelial cells to release neutrophil and monocyte chemotactic activity. Am J Physiol 262, L466-471. Kumar, R.K., Herbert, C., Thomas, P.S., Wollin, L., Beume, R., Yang, M., Webb, D.C., and Foster, P.S. (2003). Inhibition of Inflammation and Remodeling by
108 Roflumilast and Dexamethasone in Murine Chronic Asthma. Journal of Pharmacology and Experimental Therapeutics 307, 349-355. Kume, H., Takai, A., Tokuno, H., and Tomita, T. (1989). Regulation of Ca2+- dependent K+-channel activity in tracheal myocytes by phosphorylation. Nature 341, 152-154. Lazzeri, N., Belvisi, M.G., Patel, H.J., Yacoub, M.H., Chung, K.F., and Mitchell, J.A. (2001). Effects of prostaglandin E2 and cAMP elevating drugs on GM-CSF release by cultured human airway smooth muscle cells. Relevance to asthma therapy. Am J Respir Cell Mol Biol 24, 44-48. Lee, L., Liu, J., Manuel, J., and Gorczynski, R.M. (2006). A role for the immunomodulatory molecules CD200 and CD200R in regulating bone formation. Immunol Lett 105, 150-158. Lee, M.H., Reynisdottir, I., and Massague, J. (1995). Cloning of p57KIP2, a cyclin-dependent kinase inhibitor with unique domain structure and tissue distribution. Genes Dev 9, 639-649. Li, Y., Chi, L., Stechschulte, D.J., and Dileepan, K.N. (2001). Histamine-induced production of interleukin-6 and interleukin-8 by human coronary artery endothelial cells is enhanced by endotoxin and tumor necrosis factor-alpha. Microvasc Res 61, 253-262. Lipworth, B.J. (2005). Phosphodiesterase-4 inhibitors for asthma and chronic obstructive pulmonary disease. Lancet 365, 167-175. Liu, Y., Shepherd, E.G., and Nelin, L.D. (2007). MAPK phosphatases [mdash] regulating the immune response. Nat Rev Immunol 7, 202-212. Lombardi, D., Cuenoud, B., and Krämer, S.D. (2009). Lipid membrane interactions of indacaterol and salmeterol: Do they influence their pharmacological properties? European Journal of Pharmaceutical Sciences 38, 533-547. Lopez, A.D., Mathers, C.D., Ezzati, M., Jamison, D.T., and Murray, C.J. (2006). Global and regional burden of disease and risk factors, 2001: systematic analysis of population health data. Lancet 367, 1747-1757. Lynch, M.J., Baillie, G.S., and Houslay, M.D. (2007). cAMP-specific phosphodiesterase-4D5 (PDE4D5) provides a paradigm for understanding the unique non-redundant roles that PDE4 isoforms play in shaping compartmentalized cAMP cell signalling. Biochem Soc Trans 35, 938-941. Mak, J.C., Nishikawa, M., and Barnes, P.J. (1995). Glucocorticosteroids increase beta 2-adrenergic receptor transcription in human lung. Am J Physiol 268, L41- 46. Manganiello, V.C., Taira, M., Degerman, E., and Belfrage, P. (1995). Type III cGMP-inhibited cyclic nucleotide phosphodiesterases (PDE3 gene family). Cell Signal 7, 445-455. Manning, C.D., Burman, M., Christensen, S.B., Cieslinski, L.B., Essayan, D.M., Grous, M., Torphy, T.J., and Barnette, M.S. (1999). Suppression of human inflammatory cell function by subtype-selective PDE4 inhibitors correlates with inhibition of PDE4A and PDE4B. Br J Pharmacol 128, 1393-1398. Mannino, D.M. (2002). COPD: epidemiology, prevalence, morbidity and mortality, and disease heterogeneity. Chest 121, 121S-126S.
109 Mason, R.P., Rhodes, D.G., and Herbette, L.G. (1991). Reevaluating equilibrium and kinetic binding parameters for lipophilic drugs based on a structural model for drug interaction with biological membranes. J Med Chem 34, 869-877. Mattison, S., and Christensen, M. (2006). The pathophysiology of emphysema: Considerations for critical care nursing practice. Intensive and Critical Care Nursing 22, 329-337. Meja, K.K., Catley, M.C., Cambridge, L.M., Barnes, P.J., Lum, H., Newton, R., and Giembycz, M.A. (2004). Adenovirus-mediated delivery and expression of a cAMP-dependent protein kinase inhibitor gene to BEAS-2B epithelial cells abolishes the anti-inflammatory effects of rolipram, salbutamol, and prostaglandin E2: a comparison with H-89. J Pharmacol Exp Ther 309, 833-844. Milatovich, A., Bolger, G., Michaeli, T., and Francke, U. (1994). Chromosome localizations of genes for five cAMP-specific phosphodiesterases in man and mouse. Somat Cell Mol Genet 20, 75-86. Miller, A.H., Vogt, G.J., and Pearce, B.D. (2002). The phosphodiesterase type 4 inhibitor, rolipram, enhances glucocorticoid receptor function. Neuropsychopharmacology 27, 939-948. Miller, A.L., Garza, A.S., Johnson, B.H., and Thompson, E.B. (2007). Pathway interactions between MAPKs, mTOR, PKA, and the glucocorticoid receptor in lymphoid cells. Cancer Cell Int 7, 3. Mittelstadt, P.R., and Ashwell, J.D. (2001). Inhibition of AP-1 by the glucocorticoid-inducible protein GILZ. J Biol Chem 276, 29603-29610. Moodley, T., Wilson, S.M., Joshi, T., Rider, C.F., Sharma, P., Yan, D., Newton, R., and Giembycz, M.A. (2013). Phosphodiesterase 4 inhibitors augment the ability of formoterol to enhance glucocorticoid-dependent gene transcription in human airway epithelial cells: a novel mechanism for the clinical efficacy of roflumilast in severe chronic obstructive pulmonary disease. Mol Pharmacol 83, 894-906. Moulton, B.C., and Fryer, A.D. (2011). Muscarinic receptor antagonists, from folklore to pharmacology; finding drugs that actually work in asthma and COPD. Br J Pharmacol 163, 44-52. Moyer, M.L., Borror, K.C., Bona, B.J., DeFranco, D.B., and Nordeen, S.K. (1993). Modulation of cell signaling pathways can enhance or impair glucocorticoid- induced gene expression without altering the state of receptor phosphorylation. J Biol Chem 268, 22933-22940. Muise, E.S., Chute, I.C., Claveau, D., Masson, P., Boulet, L., Tkalec, L., Pon, D.J., Girard, Y., Frenette, R., and Mancini, J.A. (2002). Comparison of inhibition of ovalbumin-induced bronchoconstriction in guinea pigs and in vitro inhibition of tumor necrosis factor-α formation with phosphodiesterase 4 (PDE4) selective inhibitors. Biochemical Pharmacology 63, 1527-1535. Nannini, L.J., Lasserson, T.J., and Poole, P. (2012). Combined corticosteroid and long-acting beta(2)-agonist in one inhaler versus long-acting beta(2)-agonists for chronic obstructive pulmonary disease. Cochrane Database Syst Rev 2012. Newnham, D.M., Ingram, C.G., Mackie, A., and Lipworth, B.J. (1993). Beta- adrenoceptor subtypes mediating the airways response to BRL 35135 in man. Br J Clin Pharmacol 36, 567-571.
110 Newton, R. (2000). Molecular mechanisms of glucocorticoid action: what is important? Thorax 55, 603-613. Newton, R., and Holden, N.S. (2007). Separating transrepression and transactivation: a distressing divorce for the glucocorticoid receptor? Mol Pharmacol 72, 799-809. Newton, R., Leigh, R., and Giembycz, M.A. (2010). Pharmacological strategies for improving the efficacy and therapeutic ratio of glucocorticoids in inflammatory lung diseases. Pharmacol Ther 125, 286-327. Nicholson, C.D., and Shahid, M. (1994). Inhibitors of cyclic nucleotide phosphodiesterase isoenzymes--their potential utility in the therapy of asthma. Pulm Pharmacol 7, 1-17. Niewoehner, D.E., Erbland, M.L., Deupree, R.H., Collins, D., Gross, N.J., Light, R.W., Anderson, P., and Morgan, N.A. (1999). Effect of Systemic Glucocorticoids on Exacerbations of Chronic Obstructive Pulmonary Disease. New England Journal of Medicine 340, 1941-1947. O'Donnell, D.E., Hernandez, P., Kaplan, A., Aaron, S., Bourbeau, J., Marciniuk, D., Balter, M., Ford, G., Gervais, A., Lacasse, Y., et al. (2008). Canadian Thoracic Society recommendations for management of chronic obstructive pulmonary disease - 2008 update - highlights for primary care. Can Respir J 15, 1A-8A. Omori, K., and Kotera, J. (2007). Overview of PDEs and their regulation. Circ Res 100, 309-327. Pariante, C.M., Pearce, B.D., Pisell, T.L., Sanchez, C.I., Po, C., Su, C., and Miller, A.H. (1999). The proinflammatory cytokine, interleukin-1alpha, reduces glucocorticoid receptor translocation and function. Endocrinology 140, 4359- 4366. Pauwels, R, Buist S , Calverley, Jenkins C, and Hurd S (2001). Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Pulmonary Disease: NHLBI/WHO Global Initiative for Chronic Obstructive Lung Disease (GOLD) Workshop Summary. American Journal of Respiratory and Critical Care Medicine 163, 1256-1276. Pauwels, R.A., Lofdahl, C.G., Laitinen, L.A., Schouten, J.P., Postma, D.S., Pride, N.B., and Ohlsson, S.V. (1999). Long-term treatment with inhaled budesonide in persons with mild chronic obstructive pulmonary disease who continue smoking. European Respiratory Society Study on Chronic Obstructive Pulmonary Disease. N Engl J Med 340, 1948-1953. Peppel, K., Vinci, J.M., and Baglioni, C. (1991). The AU-rich sequences in the 3' untranslated region mediate the increased turnover of interferon mRNA induced by glucocorticoids. J Exp Med 173, 349-355. Premont, R.T. (1994). Identification of adenylyl cyclases by amplification using degenerate primers. Methods Enzymol 238, 116-127. Price, D., Chisholm, A., Ryan, D., Crockett, A., and Jones, R. (2010). The use of roflumilast in COPD: a primary care perspective. Prim Care Respir J 19, 342- 351. Profita, M., Chiappara, G., Mirabella, F., Di Giorgi, R., Chimenti, L., Costanzo, G., Riccobono, L., Bellia, V., Bousquet, J., and Vignola, A.M. (2003). Effect of
111 cilomilast (Ariflo) on TNF-α, IL-8, and GM-CSF release by airway cells of patients with COPD. Thorax 58, 573-579. Profita, M., Giorgi, R.D., Sala, A., Bonanno, A., Riccobono, L., Mirabella, F., Gjomarkaj, M., Bonsignore, G., Bousquet, J., and Vignola, A.M. (2005). Muscarinic receptors, leukotriene B4 production and neutrophilic inflammation in COPD patients. Allergy 60, 1361-1369. Pujols, L., Mullol, J., and Picado, C. (2007). Alpha and beta glucocorticoid receptors: relevance in airway diseases. Curr Allergy Asthma Rep 7, 93-99. Pujols, L., Mullol, J., Perez, M., Roca-Ferrer, J., Juan, M., Xaubet, A., Cidlowski, J.A., and Picado, C. (2001). Expression of the human glucocorticoid receptor alpha and beta isoforms in human respiratory epithelial cells and their regulation by dexamethasone. Am J Respir Cell Mol Biol 24, 49-57. Pujols, L., Xaubet, A., Ramirez, J., Mullol, J., Roca-Ferrer, J., Torrego, A., Cidlowski, J.A., and Picado, C. (2004). Expression of glucocorticoid receptors alpha and beta in steroid sensitive and steroid insensitive interstitial lung diseases. Thorax 59, 687-693. Quante, T., Ng, Y.C., Ramsay, E.E., Henness, S., Allen, J.C., Parmentier, J., Ge, Q., and Ammit, A.J. (2008). Corticosteroids reduce IL-6 in ASM cells via up- regulation of MKP-1. Am J Respir Cell Mol Biol 39, 208-217. Rabe, K., Magnussen, H., and Dent, G. (1995). Theophylline and selective PDE inhibitors as bronchodilators and smooth muscle relaxants. European Respiratory Journal 8, 637-642. Rabe, K.F., Hurd, S., Anzueto, A., Barnes, P.J., Buist, S.A., Calverley, P., Fukuchi, Y., Jenkins, C., Rodriguez-Roisin, R., van Weel, C., and Zielinski, J. (2007). Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Pulmonary Disease. American Journal of Respiratory and Critical Care Medicine 176, 532-555. Rangarajan, P.N., Umesono, K., and Evans, R.M. (1992). Modulation of glucocorticoid receptor function by protein kinase A. Mol Endocrinol 6, 1451- 1457. Reif, K., and Cyster, J.G. (2000). RGS molecule expression in murine B lymphocytes and ability to down-regulate chemotaxis to lymphoid chemokines. J Immunol 164, 4720-4729. Reinhardt, R.R., Chin, E., Zhou, J., Taira, M., Murata, T., Manganiello, V.C., and Bondy, C.A. (1995). Distinctive anatomical patterns of gene expression for cGMP-inhibited cyclic nucleotide phosphodiesterases. J Clin Invest 95, 1528- 1538. Rider, C.F., King, E.M., Holden, N.S., Giembycz, M.A., and Newton, R. (2011). Inflammatory stimuli inhibit glucocorticoid-dependent transactivation in human pulmonary epithelial cells: rescue by long-acting beta2-adrenoceptor agonists. J Pharmacol Exp Ther 338, 860-869. Rincón, M., and Davis, R.J. (2009). Regulation of the immune response by stress-activated protein kinases. Immunological Reviews 228, 212-224. Robicsek, S.A., Blanchard, D.K., Djeu, J.Y., Krzanowski, J.J., Szentivanyi, A., and Polson, J.B. (1991). Multiple high-affinity cAMP-phosphodiesterases in human T-lymphocytes. Biochem Pharmacol 42, 869-877.
112 Roffel, A.F., Elzinga, C.R., and Zaagsma, J. (1990). Muscarinic M3 receptors mediate contraction of human central and peripheral airway smooth muscle. Pulm Pharmacol 3, 47-51. Roscioni, S.S., Maarsingh, H., Elzinga, C.R., Schuur, J., Menzen, M., Halayko, A.J., Meurs, H., and Schmidt, M. (2011). Epac as a novel effector of airway smooth muscle relaxation. J Cell Mol Med 15, 1551-1563. Roth, M., Johnson, P.R., Rudiger, J.J., King, G.G., Ge, Q., Burgess, J.K., Anderson, G., Tamm, M., and Black, J.L. (2002). Interaction between glucocorticoids and beta2 agonists on bronchial airway smooth muscle cells through synchronised cellular signalling. Lancet 360, 1293-1299. Samuelsson, M.K., Pazirandeh, A., Davani, B., and Okret, S. (1999). p57Kip2, a glucocorticoid-induced inhibitor of cell cycle progression in HeLa cells. Mol Endocrinol 13, 1811-1822. Schmidt, D.T., Watson, N., Dent, G., Ruhlmann, E., Branscheid, D., Magnussen, H., and Rabe, K.F. (2000). The effect of selective and non-selective phosphodiesterase inhibitors on allergen- and leukotriene C(4)-induced contractions in passively sensitized human airways. Br J Pharmacol 131, 1607- 1618. Schudt, C, Tenor H, Loos U, Mallmann P, Szamel M, and K., R. (1993). Effects of selective PDE inhibitors on activation of human macrophages and lymphocytes. Eur Respir J 6: 367S. Sears, M.R., and Lotvall, J. (2005). Past, present and future--beta2-adrenoceptor agonists in asthma management. Respir Med 99, 152-170. Seldon, P.M., Barnes, P.J., Meja, K., and Giembycz, M.A. (1995). Suppression of lipopolysaccharide-induced tumor necrosis factor-alpha generation from human peripheral blood monocytes by inhibitors of phosphodiesterase 4: interaction with stimulants of adenylyl cyclase. Mol Pharmacol 48, 747-757. Seybold, J., Newton, R., Wright, L., Finney, P.A., Suttorp, N., Barnes, P.J., Adcock, I.M., and Giembycz, M.A. (1998). Induction of phosphodiesterases 3B, 4A4, 4D1, 4D2, and 4D3 in Jurkat T-cells and in human peripheral blood T- lymphocytes by 8-bromo-cAMP and Gs-coupled receptor agonists. Potential role in beta2-adrenoreceptor desensitization. J Biol Chem 273, 20575-20588. Shi, G.X., Harrison, K., Han, S.B., Moratz, C., and Kehrl, J.H. (2004). Toll-like receptor signaling alters the expression of regulator of G protein signaling proteins in dendritic cells: implications for G protein-coupled receptor signaling. J Immunol 172, 5175-5184. Shore, S.A., and Moore, P.E. (2003). Regulation of beta-adrenergic responses in airway smooth muscle. Respir Physiol Neurobiol 137, 179-195. Shrewsbury, S., Pyke, S., and Britton, M. (2000). Meta-analysis of increased dose of inhaled steroid or addition of salmeterol in symptomatic asthma (MIASMA). Bmj 320, 1368-1373. Silverman, E.K., Chapman, H.A., Drazen, J.M., Weiss, S.T., Rosner, B., Campbell, E.J., O'Donnell, W.J., Reilly, J.J., Ginns, L.E.O., Mentzer, S., et al. (1998). Genetic Epidemiology of Severe, Early-onset Chronic Obstructive Pulmonary Disease. American Journal of Respiratory and Critical Care Medicine 157, 1770-1778.
113 Silvestri, M., Fregonese, L., Sabatini, F., Dasic, G., and Rossi, G.A. (2001). Fluticasone and salmeterol downregulate in vitro, fibroblast proliferation and ICAM-1 or H-CAM expression. Eur Respir J 18, 139-145. Smirnov, A.N. (2002). Nuclear receptors: nomenclature, ligands, mechanisms of their effects on gene expression. Biochemistry 67, 957-977. Snelgrove, R.J., Goulding, J., Didierlaurent, A.M., Lyonga, D., Vekaria, S., Edwards, L., Gwyer, E., Sedgwick, J.D., Barclay, A.N., and Hussell, T. (2008). A critical function for CD200 in lung immune homeostasis and the severity of influenza infection. Nat Immunol 9, 1074-1083. Song, M.M., and Shuai, K. (1998). The Suppressor of Cytokine Signaling (SOCS) 1 and SOCS3 but Not SOCS2 Proteins Inhibit Interferon-mediated Antiviral and Antiproliferative Activities. Journal of Biological Chemistry 273, 35056-35062. Song, Y., Altarejos, J., Goodarzi, M.O., Inoue, H., Guo, X., Berdeaux, R., Kim, J.- H., Goode, J., Igata, M., Paz, J.C., et al. (2010). CRTC3 links catecholamine signalling to energy balance. Nature 468, 933-939. Spina, D. (2008). PDE4 inhibitors: current status. British Journal of Pharmacology 155, 308-315. Stahn, C., Lowenberg, M., Hommes, D.W., and Buttgereit, F. (2007). Molecular mechanisms of glucocorticoid action and selective glucocorticoid receptor agonists. Mol Cell Endocrinol 275, 71-78. Starr, D.B., Matsui, W., Thomas, J.R., and Yamamoto, K.R. (1996). Intracellular receptors use a common mechanism to interpret signaling information at response elements. Genes Dev 10, 1271-1283. Stockley, R.A. (1995). The pathogenesis of chronic obstructive lung diseases: implications for therapy. Qjm 88, 141-146. Sullivan, M., Olsen, A.S., and Houslay, M.D. (1999). Genomic organisation of the human cyclic AMP-specific phosphodiesterase PDE4C gene and its chromosomal localisation to 19p13.1, between RAB3A and JUND. Cell Signal 11, 735-742. Sullivan, P., Bekir, S., Jaffar, Z., Page, C., Jeffery, P., and Costello, J. (1994). Anti-inflammatory effects of low-dose oral theophylline in atopic asthma. Lancet 343, 1006-1008. Supattapone, S., Danoff, S.K., Theibert, A., Joseph, S.K., Steiner, J., and Snyder, S.H. (1988). Cyclic AMP-dependent phosphorylation of a brain inositol trisphosphate receptor decreases its release of calcium. Proc Natl Acad Sci U S A 85, 8747-8750. Szafranski, W., Cukier, A., Ramirez, A., Menga, G., Sansores, R., Nahabedian, S., Peterson, S., and Olsson, H. (2003). Efficacy and safety of budesonide/formoterol in the management of chronic obstructive pulmonary disease. European Respiratory Journal 21, 74-81. Szpirer, C., Szpirer, J., Riviere, M., Swinnen, J., Vicini, E., and Conti, M. (1995). Chromosomal localization of the human and rat genes (PDE4D and PDE4B) encoding the cAMP-specific phosphodiesterases 3 and 4. Cytogenet Cell Genet 69, 11-14.
114 Tashkin, D., and Fabbri, L. (2010). Long-acting beta-agonists in the management of chronic obstructive pulmonary disease: current and future agents. Respiratory Research 11, 149. Tashkin, D.P., Celli, B., Senn, S., Burkhart, D., Kesten, S., Menjoge, S., and Decramer, M. (2008). A 4-year trial of tiotropium in chronic obstructive pulmonary disease. N Engl J Med 359, 1543-1554. Tertyshnikova, S., and Fein, A. (1998). Inhibition of inositol 1,4,5-trisphosphate- induced Ca2+ release by cAMP-dependent protein kinase in a living cell. Proc Natl Acad Sci U S A 95, 1613-1617. Thurlbeck, W.M. (1990). Pathology of Chronic Airflow Obstruction. Chest 97, 6S- 10S. Torphy, T.J., Undem, B.J., Cieslinski, L.B., Luttmann, M.A., Reeves, M.L., and Hay, D.W. (1993). Identification, characterization and functional role of phosphodiesterase isozymes in human airway smooth muscle. J Pharmacol Exp Ther 265, 1213-1223. Traves, S.L., Smith, S.J., Barnes, P.J., and Donnelly, L.E. (2004). Specific CXC but not CC chemokines cause elevated monocyte migration in COPD: a role for CXCR2. Journal of Leukocyte Biology 76, 441-450. Trupin, L., Earnest, G., San Pedro, M., Balmes, J.R., Eisner, M.D., Yelin, E., Katz, P.P., and Blanc, P.D. (2003). The occupational burden of chronic obstructive pulmonary disease. Eur Respir J 22, 462-469. Tsingotjidou, A., Nervina, J.M., Pham, L., Bezouglaia, O., and Tetradis, S. (2002). Parathyroid hormone induces RGS-2 expression by a cyclic adenosine 3',5'-monophosphate-mediated pathway in primary neonatal murine osteoblasts. Bone 30, 677-684. Velasco, G., Armstrong, C., Morrice, N., Frame, S., and Cohen, P. (2002). Phosphorylation of the regulatory subunit of smooth muscle protein phosphatase 1M at Thr850 induces its dissociation from myosin. FEBS Lett 527, 101-104. Vestbo, J. (2004). The TORCH (towards a revolution in COPD health) survival study protocol. Eur Respir J 24, 206-210. Vignola, A.M. (2004). PDE4 inhibitors in COPD--a more selective approach to treatment. Respir Med 98, 495-503. Vine, M.M., Elliott, S.J., Haynes, M., Latycheva, O., and Hampson, C. (2010). Assessing a community-based asthma education intervention. Allergy, Asthma & Clinical Immunology C7 - P9 6, 1-1. Volonaki, E., Psarras, S., Xepapadaki, P., Psomali, D., Gourgiotis, D., and Papadopoulos, N.G. (2006). Synergistic effects of fluticasone propionate and salmeterol on inhibiting rhinovirus-induced epithelial production of remodelling- associated growth factors. Clin Exp Allergy 36, 1268-1273. Vossler, M.R., Yao, H., York, R.D., Pan, M.G., Rim, C.S., and Stork, P.J. (1997). cAMP activates MAP kinase and Elk-1 through a B-Raf- and Rap1-dependent pathway. Cell 89, 73-82. Wang, Z.Q., Xing, W.M., Fan, H.H., Wang, K.S., Zhang, H.K., Wang, Q.W., Qi, J., Yang, H.M., Yang, J., Ren, Y.N., et al. (2009). The novel lipopolysaccharide- binding protein CRISPLD2 is a critical serum protein to regulate endotoxin function. J Immunol 183, 6646-6656.
115 Ward, J.K., Barnes, P.J., Tadjkarimi, S., Yacoub, M.H., and Belvisi, M.G. (1995). Evidence for the involvement of cGMP in neural bronchodilator responses in humal trachea. The Journal of Physiology 483, 525-536. Watanabe, H., Pan, Z.Q., Schreiber-Agus, N., DePinho, R.A., Hurwitz, J., and Xiong, Y. (1998). Suppression of cell transformation by the cyclin-dependent kinase inhibitor p57KIP2 requires binding to proliferating cell nuclear antigen. Proc Natl Acad Sci U S A 95, 1392-1397. Webber, S.E., and Stock, M.J. (1992). Evidence for an atypical, or beta 3- adrenoceptor in ferret tracheal epithelium. Br J Pharmacol 105, 857-862. Wechsler, J., Choi, Y.-H., Krall, J., Ahmad, F., Manganiello, V.C., and Movsesian, M.A. (2002). Isoforms of Cyclic Nucleotide Phosphodiesterase PDE3A in Cardiac Myocytes. Journal of Biological Chemistry 277, 38072-38078. Wessler, I., Kirkpatrick, C.J., and Racke, K. (1998). Non-neuronal acetylcholine, a locally acting molecule, widely distributed in biological systems: expression and function in humans. Pharmacol Ther 77, 59-79. Wessler, I.K., and Kirkpatrick, C.J. (2001). The Non-neuronal cholinergic system: an emerging drug target in the airways. Pulm Pharmacol Ther 14, 423-434. Wilson, S.M., Sheddan, N.A., Newton, R., and Giembycz, M.A. (2011). Evidence for a second receptor for prostacyclin on human airway epithelial cells that mediates inhibition of CXCL9 and CXCL10 release. Mol Pharmacol 79, 586-595. Wollin, L., Marx, D., Wohlsen, A., and Beume, R. (2005). Roflumilast inhibition of pulmonary leukotriene production and bronchoconstriction in ovalbumin- sensitized and -challenged Guinea pigs. J Asthma 42, 873-878. Woolcock, A., Lundback, B., Ringdal, N., and Jacques, L.A. (1996). Comparison of addition of salmeterol to inhaled steroids with doubling of the dose of inhaled steroids. Am J Respir Crit Care Med 153, 1481-1488. Wright, L.C., Seybold, J., Robichaud, A., Adcock, I.M., and Barnes, P.J. (1998). Phosphodiesterase expression in human epithelial cells. Am J Physiol 275, L694- 700. Xie, Y., Jiang, H., Nguyen, H., Jia, S., Berro, A., Panettieri, R.A., Jr., Wolff, D.W., Abel, P.W., Casale, T.B., and Tu, Y. (2012). Regulator of G protein signaling 2 is a key modulator of airway hyperresponsiveness. J Allergy Clin Immunol 130, 968-976. Xie, Z., Liu, D., Liu, S., Calderon, L., Zhao, G., Turk, J., and Guo, Z. (2011). Identification of a cAMP-response element in the regulator of G-protein signaling- 2 (RGS2) promoter as a key cis-regulatory element for RGS2 transcriptional regulation by angiotensin II in cultured vascular smooth muscles. J Biol Chem 286, 44646-44658. Xu, D., Isaacs, C., Hall, I.P., and Emala, C.W. (2001). Human airway smooth muscle expresses 7 isoforms of adenylyl cyclase: a dominant role for isoform V. Am J Physiol Lung Cell Mol Physiol 281, L832-843. Xu, R.X., Hassell, A.M., Vanderwall, D., Lambert, M.H., Holmes, W.D., Luther, M.A., Rocque, W.J., Milburn, M.V., Zhao, Y., Ke, H., and Nolte, R.T. (2000). Atomic structure of PDE4: insights into phosphodiesterase mechanism and specificity. Science 288, 1822-1825.
116 Yasui, K., Agematsu, K., Shinozaki, K., Hokibara, S., Nagumo, H., Nakazawa, T., and Komiyama, A. (2000). Theophylline induces neutrophil apoptosis through adenosine A2A receptor antagonism. J Leukoc Biol 67, 529-535. Yasukawa, H., Misawa, H., Sakamoto, H., Masuhara, M., Sasaki, A., Wakioka, T., Ohtsuka, S., Imaizumi, T., Matsuda, T., Ihle, J.N., and Yoshimura, A. (1999). The JAK-binding protein JAB inhibits Janus tyrosine kinase activity through binding in the activation loop. Embo J 18, 1309-1320. Yeagley, D., and Quinn, P.G. (2005). 3',5'-cyclic adenosine monophosphate response element-binding protein and CCAAT enhancer-binding protein are dispensable for insulin inhibition of phosphoenolpyruvate carboxykinase transcription and for its synergistic induction by protein kinase A and glucocorticoids. Mol Endocrinol 19, 913-924. Yun, C.B., Lee, B.H., and Jang, T.J. (2002). Expression of glucocorticoid receptors and cyclooxygenase-2 in nasal polyps from nonallergic patients. Ann Otol Rhinol Laryngol 111, 61-67. Zhang, K.Y., Card, G.L., Suzuki, Y., Artis, D.R., Fong, D., Gillette, S., Hsieh, D., Neiman, J., West, B.L., Zhang, C., et al. (2004). A glutamine switch mechanism for nucleotide selectivity by phosphodiesterases. Mol Cell 15, 279-286. Zhu, Y., Yao, J., Meng, Y., Kasai, A., Hiramatsu, N., Hayakawa, K., Miida, T., Takeda, M., Okada, M., and Kitamura, M. (2006). Profiling of functional phosphodiesterase in mesangial cells using a CRE-SEAP-based reporting system. Br J Pharmacol 148, 833-844.
117