Transcriptional Regulation of Inflammatory Genes ... - IngentaConnect

Current Pharmaceutical Design, 2011, 17, 653-666

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Transcriptional Regulation of Inflammatory Genes Associated with Severe Asthma Rachel L. Clifford, William R. Coward, Alan J. Knox and Alison E. John Institution: Division of Respiratory Medicine and Nottingham Biomedical Research Unit, Clinical Sciences Building, City Hospital, Hucknall Road, Nottingham NG5 1PB, England, UK Abstract: The 10% of patients with the most severe asthma are responsible for a large part of healthcare expenditure and morbidity. Understanding the processes involved is key if new therapeutic approaches are to be developed. Evidence is accumulating that chronic diseases such as asthma are associated with temporal and spatial alterations in the pattern of inflammatory gene expression within the airways. Expression of these genes can be regulated by transcriptional, posttranscriptional, translational and epigenetic mechanisms. It is well established that binding of activated transcription factors to specific inducible gene promoter sites is tightly controlled by chromatin state as a result of histone modifications, particularly the balance between histone acetylation and deacetylation [1]. The interaction between transcription factors and the promoter is key to the diversification of gene expression in a time dependent manner leading to altered gene expression profiles. Alterations of the accessibility of transcription factors to the DNA can have residing effects upon gene transcription. This review will focus on the regulation of several groups of key genes which are involved in chronic airway inflammation and remodelling in asthma drawing mainly from our experience of studying these processes in airway smooth muscle cells. An overview is shown in Fig. 1.

Keywords: Transcription, asthma, smooth muscle, epigenetic, chromatin, prostanoids, chemokines, growth factors. 1. INTRODUCTION 1.1. Mechanisms of Chromatin Remodelling Chromatin is far more than an architecturally static carrier of the genetic information encoded in DNA, it also actively mediates dynamic changes in gene function and expression [2]. In quiescent cells, DNA packaging is tightly condensed to limit transcription factor accessibility. Chromatin is mainly composed of four core histones, an H3/H4 tetramer and two H2A/H2B dimers. Nucleosomes are the basic repeating units that constitute the eukaryotic chromatin, and consist of an octamer composed of two molecules each of the core histones H2A, H2B, H3 and H4 of which, 147 bp of DNA is wrapped [3]. When cells are stimulated with extracellular mediators, histones in the chromatin undergo an array of posttranscriptional modifications on their N-terminal tail domains; acetylation, phosphorylation, sumoylation, ubiquination and methylation. Histone modification patterns have been associated with distinct chromatin states and are proposed to represent a histone code that could extend the information potential of the genetic code [4], and have been closely linked to gene transcription and to the passage of epigenetic information from one cell generation to the next. Acetylation of the core histones by transcriptional coactivators with intrinsic histone acetyltransferase (HAT) activity results in changes in chromatin configuration, recruitment of cotranscriptional proteins and polymerase II which initiates transcription. In contrast, deacetylation of the core histones is generally associated with transcriptional repression [5]. Under normal physiological conditions chromatin acetylation is regulated by the balance between HATs and deacetylases (HDACs), which is essential for the maintenance of normal cellular functions. A shift in this balance has dramatic consequences on gene transcription and cellular phenotype [3]. 1.2. Histone acetyltransferases (HATs) and deacetylases (HDACs) Several transcription co-activators, including p300/CBP (CREB binding protein), PCAF (P300/CBP-associated factor) and GCN5 *Address correspondence to this author at the Institution: Division of Respiratory Medicine and Nottingham Biomedical Research Unit, Clinical Sciences Building, City Hospital, Hucknall Road, Nottingham NG5 1PB, England, UK; Tel: 0044 115 823 1713; Fax: 0044 115 823 1946; E-mail: [email protected] 1381-6128/11 $58.00+.00

(General Control Non derepressible 5), have been found to possess intrinsic HAT activity (Reviewed in [6]). HATs are divided into five families, these include the Gcn5 related acetyltransferases (GNATS), the MYST (MOZ, Ybf2/Sas3, Sas2 and Tip60) related HATs, p300/CBP HATs, the general transcription HATs which include the TFIID subunit TAF250 (TBP associated factor 250 kDa); and the nuclear hormone related HATs SRC1 (steroid receptor coactivator 1) and ACTR (activator of retinoid receptor) [7]. Most HATs exist as multisubunit complexes, displaying distinct substrate specificities, with associated subunits regulating the activity of the respective catalytic subunits [8]. Recruitment of a histone modifying enzyme to a gene promoter is not specific in it self however the specificity of the enzyme for specific histone residue will produce a specific biological outcome. Gcn5 and PCAF preferentially acetylate H3K9 and K14 whereas NuA4 complexes preferentially acetylate K4, K8, K12 and K16 of H4 [9]. HDACs play a critical role in reversing the hyperacetylation of core histones. HDACs have been described in three classes according to their structures, expression patterns and catalytic mechanisms. Class I comprising HDAC1, 2, 3, and 8 with molecular weights of 22-55 kDa with homology in their catalytic sites. Class II HDACs include HDAC4, 5, 6, 7, 9 and 10, are larger molecules with molecular weights between 120 kDa and 135 kDa. A subclass of Class II includes, class IIa which includes HDAC6 and 10 containing two catalytic sites. Class III HDACs (Sirt1, 2, 3, 4, 5, 6, and 7) do not have histones as a primary target. HDAC 11 contains a conserved domain in the catalytic region of both Class I and Class II and is grouped to Class IV. These HDAC classes are not redundant in their biological activity. Class I HDACs are expressed primarily in the nucleus being involved in gene silencing of both specific genes and entire chromosomal domains and play a role in cell proliferation. In contrast Class II HDACs possess the capability of active nucleo-cytoplasmic shuttling, which has been suggested to be regulated by various signalling molecules and cell specific regulation and have a more tissue specific function [10-12]. HDAC complexes are generally recruited to transcription factors by ‘‘bridging’’ factors; silencing mediator of retinoid and thyroid receptor (SMRT), Co-RE1 silencing transcription factor (CoREST) [13, 14], switch independent 3 (mSin3), nuclear receptor co-repressor (NCoR), nucleosome remodelling and decatylase (NuRD). These co-repressor complexes regulate gene repression and may provide specificity by selecting which genes are repressed [15-17]. HDAC1 © 2011 Bentham Science Publishers Ltd.

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NCoR/CoRest A

HMTs

HDACs

Me

Me

Me

X

Transcription inhibited by closed chromatin structure K9/K27 Methylation

Me

Me

Me

Acetylation Cytokine stimulation (eg IL-1, TNF, TGF)

HDMs

B

Me X

Ac

HATs

Histone modification

Ac

Ac

K9/K27 Methylation Ac

Ac

Ac

Acetylation

C

Chromatin remodelling Ac

Ac

Ac

Ac

Ac

Ac

D Ac

Ac

Recruitment of transcription factors , co-factors, RNA Pol II and elongation factors

Initiation of transcription

Ac

mRNA Ac

Ac

Ac

Fig. (1). Schematic representation of the transcriptional regulation of gene expression. A) In quiescent cells, DNA packaging is tightly condensed to limit transcription factor accessibility. Multi protein repression complexes (NCoR/CoREST) which contain histone deacetylases (HDACs) are associated with the DNA and maintain hypoacteylation of core histones. In addition, histone methyltransferases (HMT) facilitate methylation of specific histone lysine residues (K9 and K27) resulting in repression of transcription. B) Following cytokine stimulation, transcriptional repression is lost due to demethylation (HDMs) of repressive lysine residues, recruitment of histone acetylases (HATS) and subsequent lysine acetylation. C) The loss of repression leads to chromatin remodeling, loosening of the DNA packaging and increased accessibility for transcription factors (TF), co-factors and RNA polymerase (Pol) II. D) The correct chromatin environment allows association of gene specific transcriptional activators and successful transcription of mRNA. HDM, histone demethylase.

and HDAC2 exist together in at least three multi protein complexes; Sin3, NuRD and CoREST [18, 19] and NCoR exist in core repression complexes with HDAC3. HDACs can be inhibited by a range of inhibitors divided into four groups based upon their structure; hydroximates, cyclic peptides, aliphatic acids and benzamides. Trichostatin A (TSA), the first natural product discovered, and its structural analog suberoyl anilide hydroxamic acid (SAHA) belong to the hydroximates group and inhibit the activity of Class I and 2 HDACs [20]. HDAC inhibitors are thought to interact with the catalytic domain of HDACs to block the substrate recognition ability of these HDACs [21]. Class I and II HDAC family members are zinc binding enzymes, and are inhibited by the HDAC inhibitor SAHA. Class III HDACs are Zn2+ independent and NAD dependent and are not inhibited by SAHA. Results of treatment with HDAC inhibitors in clinical studies have shown significant anticancer ac-

tivity [22] but may not prove as successful in other disease pathologies due to a lack of potency and detrimental cytotoxicity with long term therapy [23]. Many human diseases, particularly cancer, have an epigenetic aetiology; drugs targeting these processes have been applied as novel therapies. It is unlikely that aberrant regulation of a single gene can explain the etiology of asthma but the knowledge gained from analysis of gene expression may serve as a basis for the identification of biomarkers for the detection of disease and the development of effective therapies. 1.3. Smooth Muscle Cells Although altered patterns of gene expression and inflammatory mediator release from multiple cell types appear to contribute to the pathogenesis of asthma, airway smooth muscle (ASM) cells appear to play a key role in the disease. Post mortem studies from asth-

Transcriptional Regulation of Inflammatory Genes

matic airways have demonstrated thickening of the airway smooth muscle layer [24, 25]. The increased smooth muscle layer is thought to contribute to airway narrowing and bronchial hyperresponsiveness through the geometric effects of airway thickening and by the thickened muscle developing an exaggerated shortening response to bronchoconstrictor stimuli [26]. Our understanding of airway smooth muscle functions in asthma has advanced considerably over the last 10-15 years. The traditional view of airway smooth muscle in asthma is of a passive partner in airway inflammation, contracting in response to proinflammatory mediators and neurotransmitters and relaxing in response to endogenous and exogenous bronchodilators [27]. This paradigm was overly simplistic and it is clear that ASM has several other important properties of relevance to obstructive lung diseases. These functions include the ability to proliferate, undergo hypertrophy and migrate and thereby contribute to the dysfunctional repair mechanisms that cause airway remodelling and poorly reversible airflow obstruction. Of particular note are its synthetic functions whereby ASM cells synthesise and release a diverse repertoire of biologically active inflammatory mediators. ASM is now considered to play a central role in orchestrating the inflammatory response within the bronchial wall. In this chapter we will discuss some of the key molecules that these cells produce and the molecular mechanisms responsible. We have focussed on ASM in view of its central importance in asthma but a number of the same regulatory mechanisms also operate in other structural cells such as epithelium and fibroblasts. 2. PROSTANOIDS 2.1. Prostanoid Production from Smooth Muscle Cells and their Involvement in Asthmatic Airways Eicosanoids are important signalling molecules which are produced locally both physiologically and during inflammatory disease and have potent effects on a number of inflammatory processes. There are four families of eicosanoids; the prostaglandins, prostacyclins, thromboxanes and leukotrienes. Two families of enzymes catalyze fatty acid oxygenation to produce the eicosanoids: Cyclooxygenase (prostaglandin H synthase) which generates the prostanoids and Lipoxygenase, 5-lipoxygenase generates the leukotrienes [28]. The enzyme 5-lipoxygenase (5-LO) uses 5lipoxygenase activating protein (FLAP) to convert arachidonic acid into 5-hydroperoxyeicosatetraenoic acid (5-HPETE), which spontaneously reduces to 5-hydroxyeicosatetraenoic acid (5-HETE). The enzyme 5-LO acts again on 5-HETE to convert it into leukotriene A4 (LTA4), which may be converted into LTB4 by the enzyme leukotriene A4 epoxide hydrolase. The leukotrienes LTC4, LTD4 and LTE4 all contain cysteine and are collectively known as the cysteinyl leukotrienes. The prostaglandins are produced from endogenous arachidonic acid via the cyclooxygenase (COX) pathway. This is a two step process involving cyclooxygenase activity to convert arachidonic acid to PGG2 and a peroxidase reaction to produce PGH2. PGH2 can thereafter be converted to thromboxanes or to prostaglandins PGD2, PGF2, PGE2 and PGI2 by specific synthases or isomerases. Cyclooxygenase appear to be in the majority of cells, the cyclooxygenase metabolites released from specific cells reflects the arsenal of isomerases and synthases and will ultimately determine whether a cell produces a protective or proinflammatory prostanoid response to an inflammatory insult. Airway smooth muscle is a rich source of prostaglandins with PGE2 and to a lesser extent PGI2 being the main prostanoids produced [28] [29-31]. Prostanoids have varied effects on airway smooth muscle tone, both PGD2 and PGF2 contract human airway smooth muscle as does thromboxane A2 [32]. PGD2 and PGF2 are potent bronchoconstrictors of human airways [33], sub threshold concentrations of PGD2 also increase airway responsiveness to other agents [34]. In contrast PGE2 relaxes airway smooth muscle at

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low concentrations and contracts at higher concentrations [32] and is potentially able to inhibit induced bronchoconstriction [35, 36] and can be thought of protective in the lung. As will be discussed below, induction of COX-2 and subsequent PGE2 is an important autocrine loop in the induction of chemokines and growth factors in human airway smooth muscle cells in response to some stimuli but not others. For example bradykinin induces IL-8 induction via the induction of COX-2 derived PGE2 [37]. In addition IL-1 induced PGE2 facilitates G-CSF and GM-CSF secretion [38, 39]. PGE2 also increases VEGF [40, 41] and more recently we have demonstrated that Endothelin-1 induces the expression of Amphiregulin, Follistatin, Inhibin--A and Epiregulin via a COX-2 dependent PGI2 synthesis [42]. 2.2. Consequences of Modulating Prostanoid Production in Asthmatic Smooth Muscle Asthma is characterised by non specific bronchial hyperresponsiveness to a variety of stimuli [27, 43]. It is not clear whether the consequence of COX-2 induction and the resultant prostaglandin production in asthma is deleterious or beneficial. PGE2 is an important anti inflammatory mediator and has considerable bronchoprotective effects in the airway [36]. PGE2 also inhibits human airway smooth muscle proliferation [44] and collagen synthesis in fibroblasts [45]. High concentrations of PGE2, however can cause airway smooth muscle contraction leading to a potential deleterious effect of COX-2 induction in asthma [32]. There is scant evidence to support the view that airway smooth muscle contraction derived from asthmatic patients is abnormal; the evidence that 2 adrenoceptor linked relaxant mechanisms maybe dysfunctional in asthmatic airways is more compelling [46-49], and that this effect is mediated, in part, by IL-1 and Bradykinin mediated induction of COX-2. [50-52]. The induction of COX-2 is associated with PGE 2 and PGI2, which are both coupled to adenyl cyclase and therefore intracellular cAMP. The elevated levels of PGE2 and PGI2 cause heterologous desensitization of adenyly cyclase, thereby impairing cAMP generation in response to 2 adrenoceptor agonists [31]. This may suggest that up regulation of COX-2 as a result of airway inflammation in asthma, impairs the actions of 2 adrenoreceptor agonists used to treat bronchoconstriction. By preventing COX-2 induction, it may be possible to restore 2 adrenergic responsiveness. The effects of cyclooxygenase inhibitors on bronchoconstriction depend upon the inhibitor and the route of administration. Indomethacin can inhibit refractoriness and the bronchoprotective effects of frusemide [36] suggesting that under circumstances where protective prostaglandins such as PGE2 are produced these drugs have a deleterious effect. This effect contrasts with the effects during induced bronchoconstriction where the balance of prostanoid production has presumably shifted to proinflammatory prostanoids. In addition the use of aspirin has been demonstrated to exacerbate asthma in a subset of patients which increased amounts of leukotrienes [53]. Aspirin induced bronchoconstriction is thought to be caused by shutting off arachidonic acid metabolism away from prostanoid production to leukotriene production and bronchoconstriction which can be inhibited in these patients with leukotriene antagonists [54]. Induction of COX-2 can be inhibited by the glucocorticoid dexamethasone. The most likely mechanism for this effect is via the inhibition of NF-B [55]. The effect of COX inhibitors in asthma is likely to depend upon the balance between proinflammatory PGD2, PGF2, thromboxane A2 and anti inflammatory PGE2. 2.3. Molecular Regulation of COX-2 Transcription Cyclooxygenase is the rate limiting enzyme for the conversion of arachidonic acid to prostanoids and exists in two isoforms. COX1 is constitutively expressed in airway smooth muscle whereas COX-2 is induced by mediators and cytokines present in the inflammatory milieu including IL-1 and bradykinin [56-58] with TNF- and IFN- having little effect [57]. These isoforms are the


products of distinct genes. The gene for COX-1 is situated on chromosome 9 whereas the gene for COX-2 is on chromosome 1 [59]. There are several differences between COX-1 and COX-2; the introns of COX-1 encompass 22kb compared with 8kb for COX-2. COX-1 contains two control elements for SP-1 in its promoter and the 3’-UTR has 2 polyadenylation sites giving rise to 2.8 and 5.2kb isoforms. The COX-1 3’ UTR also contains a single AUUUA mRNA instability motif and no TATA box in its promoter therefore produces a continuous transcribed stable message [59]. COX-2 is an immediate early gene and its expression is subject to multilevel regulation through both transcriptional and posttranscriptional mechanisms. COX-2 is encoded by a 7.5-kb genomic DNA with 10 exons [60]. The 5'-flanking promoter region of human COX-2 contains a canonical TATA-box and multiple regulatory elements, including two putative nuclear factor B (NF-B)-binding sites, one nuclear factor interleukin-6 (NF-IL-6)/CCAAT/enhancer-binding protein (C/EBP)-binding site, and one cyclic AMP-response element (CRE) [61]. COX-2 expression is critically governed by different transcription factors, including CRE-binding protein (CREB) [62], C/EBP [63], activating protein-1 (AP-1) [64], and NF-B [65], in a highly cell type-specific and stimulus-specific manner. In human airway smooth muscle cells it has been demonstrated that the requirement of the cyclic AMP response element (CRE) in response to bradykinin, whereas nuclear factor IL-6 and CRE, and to a lesser extent, nuclear factor kappa B (NF-B) are involved in IL-1 induced COX-2 induction [66]. COX-2 mRNA also contains several polyadenylation signals and multiple AUUUA instability sequences that mediate rapid transcript degradation [67]. Although the enzymes are of similar size and structure the cellular location of the two isoforms differs. Both COX-1 and COX-2 are localized to the endoplasmic reticulum, whereas COX-2 is also localized to the nuclear membrane [68] which would allow it to have a role in altering gene transcription. We have previously reported that induced COX-2 gene transcription in human airway smooth muscle cells is closely associated with histone H4 acetylation at the COX-2 promoter site [66]. Transcriptional regulation of human COX-2 has been studied in other cell systems demonstrating it to be a key element in various pathophysiological processes, including inflammation, cardiovascular disease, tissue remodelling, cancer [69, 70] and fibrosis [71]. Our own studies in lung fibroblasts have shown that the COX-2 promoter is regulated in a complex manner through recruitment of HATs and HDAC containing co-repressor complexes, NCOR, CoREST and Sin3a. 3. CHEMOKINES Chemokines are small chemoattractant cytokines (8-14kDa) with diverse effects on cellular recruitment, activation and differentiation via interactions with 7-transmembrane spanning G-protein coupled chemokine receptors [72]. They are classified into four subfamilies, CXC, CC, C and CX3C on the basis of their sequence homology and the position of conserved cysteine residues within the protein [73]. Chemokines have diverse effects on multiple cell types and play a critical role in the pathogenesis of acute and chronic inflammatory lung diseases [74-76]. Elevated levels of a number of chemokines have been detected in the airways of patients with asthma including CCL5/RANTES, CCL11/Eotaxin, IP10, and CXCL8/IL-8 [75-77]. These chemokines can interact with specific chemokine receptors to recruit and activate a range of leukocyte subtypes including eosinophils, TH2 lymphocytes, neutrophils and mast cells to the bronchi [78]. In addition, they are able to activate or modulate the phenotype of structural cells within the airways [79, 80]. Numerous studies have established a role for chemokines in controlling various stages of the asthma pathology and some of these chemokines, their receptors and their cellular sources are detailed in Table 1.

Clifford et al.

Although there are many cell types which contribute to elevated chemokine production within the airways of asthmatic individuals, airway smooth muscle (ASM) cells have been identified as a major source of a variety of different chemokines and we will focus primarily on these cells in this review. Following stimulation with inflammatory mediators, ASM secrete the CC chemokine family members, CCL11 [81, 82], CCL5 [83-85], CCL2, CCL7, CCL8[84, 86], CCL19[87] and CCL17 [88]; the CXC chemokines CXCL8 [86, 89, 90], CXCL10 [91, 92], CXCL1, CXCL2 and CXCL3 [93] and finally, CX3CL1 [94, 95]. In recent years, a number of studies comparing chemokine release in ASM isolated and cultured from non-asthmatic and asthmatic airways have demonstrated that asthmatic cells are hypersecretory for CCL11, CXCL10 and CXCL8. The ability of ASM to synthesise and release chemokines during an inflammatory response has profound implications for the regulation of airway inflammation and the hypersecretory phenotype of these cells in asthmatic airways suggests that they are a target for anti-inflammatory therapy with agents such as glucocorticoids and 2-agonists. A clearer understanding of the molecular mechanisms regulating chemokine expression in ASM may lead to novel therapies for asthma and as these mechanisms appear to be chemokine and stimulus-specific it is unlikely that one type of therapy will successfully target all chemokines in an inflammatory disease. Although many studies have identified ASM as a major source of chemokines in asthma, very few studies have performed any detailed analysis of the transcriptional mechanisms regulating chemokine production. Most of the data is available for the CC chemokines CCL5/ RANTES, CCL11/Eotaxin and CCL2/MCP-1, the CXC chemokines CXCL/IL-8 and CXCL10/IP-10 although some data exists for and CCL17TARC. 3.1. CC CHEMOKINES 3.1.1. CCL11 CCL11 is constitutively released by ASM but secretion is further induced by stimulation with TNF, IL-1, IL-4 and IL-13 [81, 82, 96]. The human CCL11 promoter contains C/EBP, AP-1, STAT6, and NF-B binding sites [97, 98]. Induction of CCL11 by the Th2 cytokines, IL-4 and IL-13, is mediated via STAT6 activation whereas TNF-induced transcription is dependent on NFB but independent of STAT-6 [99, 100]. TGF has recently been shown to enhance Th2 cytokine induced CCL11 production, although it is unable to directly induce CCL11 expression. The cooperative effect of TGF on IL-4-induced CCL11 expression appears to be regulated not by Smad, C/EBP or AP-1 binding, but rather by activation of NF-B. Pretreatment of HASM with the NFB inhibitor, BAY11-7085, and the glucocorticoid fluticasone propionate significantly inhibited CCL11 mRNA induction following combined IL-4/TGF stimulation suggesting a key role for NFB in the cooperative activation of CCL11 by these two cytokines. Mutation of STAT-6 binding sites was also shown to reduce both IL-4 and combined IL-4 and TGF effects on the CCL11 promoter [101]. Inflammatory cytokines also regulate chemokine gene expression by chromatin remodeling. Histone modifications regulate the unravelling of DNA and as a result transcription factor binding sites are exposed and gene transcription is initiated [102, 103]. Our recent studies have shown that Histone H4 acetylation following TNF stimulation is a key event in regulating binding of NF-B p65 to the eotaxin promoter and subsequent transcription of CCL11. This acetylation of Histone H4 is mediated by the HAT, p/caf, which is recruited to the CCL11 promoter following its phosphorylation by PKCII [104]. Other enzymes implicated in regulating CCL11 release include p38 MAP kinase, JNK kinase and p42/p44 ERK following IL1 stimulation [105, 106]. ERK activation is also involved in IL-13,


Table 1.


657

Chemokines and Chemokine Receptors Involved in Asthma

Chemokine Nomenclature

Common Name in Human

Cellular Sources in the Lung

Receptors

CCL2

MCP-1, MCAF

ASM, EP, F, Alv M,

CCR2

CCL3

MIP-1

EP

CCR1, 5

CCL5

RANTES

ASM, EP, E

CCR1, 3, 5

CCL7

MCP-3

EP

CCR1, 2, 3

CCL8

MCP-2

ASM, EP

CCR2, 3

CCL11

Eotaxin

ASM, EP, Alv M, EC, F, E

CCR3

CCL13

MCP-4

EP

CCR2, 3

CCL17

TARC

EP

CCR4, 8

CCL19

ELC, MIP-3

ASM, MC

CCR7

CCL22

MDC, STCP-1

SM, Alv M, EP

CCR4

CCL24

MPIF-2, Eotaxin-2

EP

CCR3

CCL26

Eotaxin-3

ASM, EP

CCR3

CCL28

CCL28, MEC

EP, ASM, alv M, E

CCR10

CXCL8

IL-8

ASM, EP, EC, Alv M

CXCR1, 2

CXCL9

MIG

EP

CXCR3

CXCL10

IP-10

ASM, EP, M, F

CXCR3

CXCL12

SDF-1/

EC, Alv M, T

CXCR4

Abbreviations : - Alv M Alveolar macrophage; E eosinophil; EC endothelial cell; EP epithelial cell; F fibroblast; M monocyte/macrophage; MC mast cell; ASM airway smooth muscle cell; T T-cell.

IL-4 or TNF induced eotaxin release [99]. In contrast, cAMP has been shown to inhibit IL-1b mediated CCL11 release [105, 106]. 2-agonists and glucocorticoids, partially inhibit TNF-induced eotaxin production from HASM, and as with CXCL8, their combined use results in greater inhibition of chemokine gene transcription[96]. These compounds inhibit histone H4 acetylation and the conformational change in chromatin inhibits NFB p65 binding to the eotaxin promoter [100]. 3.1.2. CCL2/MCP-1 ASM cells release CCL2/MCP-1 constitutively, although mRNA and protein levels are significantly increased in response to IL-1, TNF or Endothelin-1 [84, 86, 107, 108]. IL-1-induced CCL2 is mediated by activation of p44/42 and p38MAPK, JNK Kinase, ERK pathways and NF-B [106]. Like IL-1, ET-1 stimulation has also been shown to activate p44/42 and p38 MAPK pathways suggesting that crosstalk could occur between cytokineand GPCR-mediated pathways of chemokine expression. Sutcliffe et al [108] recently preformed a detailed analysis of the transcriptional regulation of CCL2 production by ET-1. The magnitude of ET-1’s effects on the CCL2 promoter and enhancer suggested that transcription factor binding to the promoter was more important in regulating chemokine release. The CCL2 promoter contains binding sites for AP-1, Sp-1, NF-kB and NF-1 in addition to a CAAT box. Serial deletion studies of the CCL2 promoter determined that the region of the promoter required for maximal activation following ET-1 stimulation contained consensus binding sites for NF-B and AP-1. ChIP analysis confirmed ET-1-induced binding of p65 and cjun to the CCL2 promoter and binding was inhibited by the MEK inhibitor PD98059 and the p38 MAPK inhibitor SB203580 suggesting that these kinases mediate ET-1 stimulated CCL2 transcription

via NF-B and AP-1. The importance of p38MAPK, ERK and JAK activation in CCL2 transcription has also been confirmed in epithelial cells stimulated with IL-4 and IL-13[109]. Increasing cAMP reduced MCP-1 but not via p38 MAPK, ERK or JNK inhibition [110] 3.1.3. CCL5/RANTES In ASM, CCL5 mRNA and protein is induced by TNF, IL-1 or platelet activating factor (PAF) and can be augmented by IFN [83-85]. The CCL5 promoter contains several key response elements, including a CD28-responsive element (CD28RE), two AP-1 binding sites, binding sites for signal transducer and activator of transcription (STAT) protein, nuclear factor of activated T cells (NF-AT), and C/EBP, and two NF-B binding elements [111]. Using a series of site-directed mutations within the human CCL5 promoter, Ammit and colleagues determined that AP-1 and NF-AT but not NF-B regulate activation of the CCL5 promoter [112]. Furthermore TNF-induced AP-1 DNA binding was attenuated by dexamethasone. Elevated cAMP levels also inhibit cytokineinduced RANTES secretion from ASM cells [83, 105] although not by inhibiting AP-1 DNA binding [112]. IL-1-stimulated RANTES release is dependent on JNK [113] and p42/p44 ERK activation but not p38 MAPK [114]. 3.1.4. CCL17/TARC The TH2 cell chemoattractant, CCL17 is released by HASM by combined stimulation with either IL-4 or IL-13 and TNF [88]. IL4, IL-13, IL-1, TNFA and IFN alone do not induce CCL17 release. Little is known about the molecular mechanisms regulating TARC gene expression in ASM although in bronchial epithelial cells NFB is required for TNF induced TARC release [115]. In HaCaT keratinocytes, combined TNF and IFN treatment induced


CCL17 via NF-B and p38 MAPK pathways [116]. In addition, IL4-mediated CCL17 production has also been shown to be STAT-6 dependent in T-cells[117] and macrophages[118]. In ASM, IL-4 or IL-13 and TNF induced TARC release is inhibited by isoproterenol, cAMP analogues or forskolin [88] but not dexamethasone. 3.2. CXC Chemokines 3.2.1. CXCL8/IL-8 ASM release CXCL8 in large quantities following stimulation with TNF, IL-1 [86, 119], TGF [120] or bradykinin (BK) [37, 121]. The core CXCL8 promoter contains binding motifs for the transcription factors NFB, AP-1 and C/EBP [122]. In many studies of normal human cells, NFB activation has been shown to be essential for CXCL8 transcription whilst AP-1 and C/EBP binding are dispensable for transcription in some cell types but required for promoter activation in others [122, 123]. In airway smooth muscle cells, mutation analysis of the CXCL8 promoter determined that NF-B is the major transcription factor in TNF- or BK-stimulated CXCL8 gene transcription but that AP-1 and C/EBP also contribute to maximal activation of the promoter [37, 124]. ChIP analysis indicates a temporal recruitment of these three transcription factors to the CXCL8 promoter following TNF stimulation with maximal binding of C/EBP (at 30 minutes) preceding the arrival of NFBp65 and AP-1 (60 minutes). C/EBP is known to form functional associations with members of the NF-B family including NF-Bp65, and simultaneous binding of these transcription factors in close proximity can have both synergistic and co-operative effects of CXCL8 transcription following exposure to inflammatory stimuli [125, 126]. ASM isolated from the airways of asthmatic individuals have been found to hypersecrete CXCL8 both basally and following TNF treatment and this increased chemokine production results from an abnormality in the transcriptional control of CXCL8 [124]. The asthmatic ASM show no global increase in NFB activity but do show a selective increase in NF-Bp65 and C/EBP binding along with increased RNA polymerase II recruitment to the CXCL8 promoter. This active transcription complex is detected in association with the promoter DNA of asthmatic cells even in the absence of cytokine stimulation. The active transcription complex appears to be responsible for the increase in mRNA synthesis in the asthmatic ASM and the hypersecretion of CXCL8. Cytokine-mediated CXCL8 release has also been shown to require activation of ERK, p38MAPK [127-129], PI3K [130] and JNK pathways [113] whilst the synergistic effects of IL-17 and IL1 on CXCL8 mRNA and protein release from ASM are mediated by ERK1/2 and p38MAPK signal transduction pathways and require the co-operation of NF-B and AP-1 cis-acting elements upstream of the CXCL8 gene[130]. Glucocorticoids suppress TNF-stimulated CXCL8 production by ASM, and 2 agonists augment their inhibition [119]. Dexamethasone inhibits BK-induced IL-8 production via the inhibition of NF-B, AP-1, and NF-IL-6 binding. IFN abrogates TNFinduced NF-B-dependent gene expression by impairing NF-B transactivation [131] via an increase in HDAC activity. 3.2.2. CXCL10/IP-10 Interferon--inducible protein-10 (IP-10) is preferentially expressed in ASM in bronchial biopsies from asthmatic and chromic obstructive pulmonary disease (COPD) patients and in ex vivo ASM cultures from asthmatic patients [91, 92]. TNF-induced CXCL10 in HASM is dependent on NF-B activation as the specific NF-B inhibitor, salicylanilide, blocked TNF but not IFN mediated expression of the chemokine. TNF has also been shown to induce CXCL10 via activation of p38MAPK. IFN activates JAK/STAT1a and inhibition of the JAK/STAT pathway abrogates CXCL10 release more effectively than the steroid fluticasone [132]. TNF and IFN synergistically enhanced IP-10 mRNA and protein

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accumulation compared with either cytokine alone [92]. The synergistic effects of combined TNF and IFN treatment on CXCL10 release in HASM are not regulated at the level of NFkB activation, STAT-1a phosphorylation or in vivo binding of these transcription factors to the CXCL10 promoter. The synergy has been found to lie at the level of transcriptional coactivator recruitment with TNF and IFN inducing a synergistic increase in binding of the CREBbinding protein (CBP) to the CXCL10 promoter which is accompanied by association of RNA Polymerase II with the promoter [132]. Previous studies have demonstrated that isolated fibroblasts from IPF patients constitutively express less IP-10, a strong inhibitor of angiogenesis, and more IL-8, thereby inducing greater angiogenic activity [133]. We revealed that the repression of IP-10 in these cells involved not only HDACs, as with cyclooxygenase-2 repression, but also histone H3 hypermethylation. Together with the HDAC containing repressor complexes, the IP-10 promoter histone was associated with the binding of the methyltransferases G9a and SUV39H1 and heterochromatin protein 1 (HP1). More importantly, treatment of diseased cells with HDAC or G9a inhibitors similarly reversed the repressive HDAC and histone hypermethylation and restored IP-10 expression. These findings strongly suggest that epigenetic dysregulation involving interactions between HDACs and histone hypermethylation is responsible for targeted repression of IP-10 and that this is amenable to therapeutic targeting [134]. 4. GROWTH FACTORS Growth factors are broadly defined as proteins that stimulate cellular growth, proliferation and differentiation. A number of the structural changes that occur in the asthmatic lung are postulated to be regulated by growth factors. For example, increased deposition of matrix and increased numbers of smooth muscle cells maybe due to the action of transforming growth factors beta (TGF) and epidermal growth factor (EGF) respectively. Vascular endothelial growth factor (VEGF) likely contributes to the increased size and number of blood vessels in the asthmatic lung [135]. Growth factors are secreted from a number of cells in the lung and their secretion is regulated by varied transcriptional mechanisms as outlined in Table 2. Furthermore they are a target for asthma therapeutics including 2 agonists and corticosteroids. 4.1. Basic Fibroblast Growth Factor (bFGF) bFGF is a heparin binding growth factor known to affect growth and differentiation of numerous cell types. It also contributes to inflammation and angiogenesis, both fundamental processes in asthma pathogenesis and airway remodelling [136]. More specifically bFGF induces airway smooth muscle (ASM) cell proliferation and migration [136]. Bronchoalveolar lavage (BAL) concentrations of bFGF are higher in asthmatic subjects than non-asthmatic controls and increase further on bronchoprovocation [137]. The main source of bFGF is via release from mast cells or from the extracellular matrix (ECM) following exposure to mast cell degranulation products including heparin. However, epithelial cells are also a source of bFGF [138] and elevated basal levels may result either from increased synthesis or reduced degradation of bFGF in asthmatic individuals [137]. In agreement with this, epithelial bFGF immunostaining is greater in asthmatic versus non-asthmatic tissue [135, 138]. Increased synthesis of bFGF in the asthmatic lung implicates transcriptional regulation of bFGF although the transcriptional mechanisms responsible have been poorly investigated. Detection of bFGF mRNA has proved troublesome, potentially due to low promoter activity and/or instable transcripts and therefore has often required the use of over-expression systems. However, increased bFGF mRNA levels are seen following tissue damage [139] and use of promoter luciferase reporters has allowed some basic transcriptional mechanisms to be elucidated.


Table 2.

659

Source and Regulation of Growth Factors Relevant to Asthma

Growth Factor bFGF


Source SM, EP and M Cells

Potential Transcription Factors Involved Egr-1 HOXB7

Drug Inhibition Budesonide (rat Ova) Salmeterol (F) Fluticasone (F)

EGF

SM and EP Cells (Receptor: - NA - EP and glandular cells A – extends to SM and basement membrane)

NFB, AP1-3, Sp1, p53, HRE, C/EBP

IGF

EP and rabbit airway SM cells (IGF-I)

IGF-I: STAT-5, SV40/p53 (+p300 and pRb), Androgen receptor IGF-II: Egr-1

Beclomethasone (IGF-I)

VEGF

SM and EP

Sp1, STAT-3, HIF1

Beclomethasone (sputum and stimulated SM) Fluticasone (Sputum and biopsy staining) Pranlukast (sputum) Formoterol/Salmeterol (EP cells)

Abbreviations: - SM smooth muscle; EP epithelial; M mast; F fibroblast; NA non-asthmatic; A asthmatic

The bFGF gene was cloned in 1986 [140] and the promoter characterised in 1991 [141] to reveal five GC boxes (putative Sp1 binding sites) and one potential AP-1 binding site. To date, Sp-1 has been shown to bind the basal bFGF promoter but with unknown consequence and is not regulated in response to stimulus [142]. In contrast, AP-1 regulates bFGF expression in response to ionizing radiation [143] and heat treatment [144] via Protein kinase C (PKC) in human breast carcinoma cells (MCF-7). Interestingly bFGF is a transcriptional autoregulator (it can induce its own transcription). In the human hepatocellular carcinoma cell line, Hep3B, autoregulation is mediated via upregulation of the transcription factor Egr-1 and binding of Egr-1 to two Egr-1 binging sites in the bFGF promoter (-165- GCGGGGGTG—157 and -63-CGCCCCGA—55)[145]. Furthermore, in astrocytes, endothelin-3 (ET-3) stimulates bFGF transcription via upregulation of Egr-1 protein and association of Egr-1 with the bFGF promoter at the -63bp binding site, while atrial natriuretic peptode (ANP) inhibites ET-3 induced bFGF via downregulation of Egr-1 [142]. Homeobox protein 7 (HOXB7) has also been shown to regulate bFGF expression. No mechanistic studies regarding increased bFGF expression in asthma have been performed, despite knowledge of bFGF secretion from airway smooth muscle [146] and epithelial cells [147]. Future studies investigating the transcriptional mechanisms of bFGF production from these cells and/or differential regulation of bFGF in asthmatic versus non-asthmatic cells would be informative. Little is known about the effects of 2-adrenoceptor agonists and/or corticosteroids on bFGF expression. Cang and Luan [148] showed the corticosteroid budesonide to reduce bFGF expression in a rat OVA model of asthma, while Silvestri et al [149] showed the 2-adrenoceptor agonist salmeterol to inhibit bFGF induced lung fibroblast proliferation. In alternative respiratory diseases untreated COPD patients have increased numbers of bFGF positively stained cells compared to control and beclomethasone treated patients [150] and fluticasone inhibits TNF- induced bFGF production from nasal polyp fibroblasts [151]. All studies are observational and elucidation of the mechanisms utilised by the drugs requires further investigation.

4.2. Epidermal Growth Factor (EGF) EGF is one of a group of ligands that signal via the EGF receptor. Other ligands include transforming growth factor (TGF)-, amphiregulin, epiregulin , neuroregulin , heparin-binding EGF and betacellulin. Receptor signalling results in an array of cellular events including growth promotion, growth inhibition, protection against apoptosis, induction of differentiation, reorganisation of the cytoskeleton and cell migration [152]. More specifically to airway remodelling, EGF is an established inducer of ASM cell proliferation [153, 154] and modulator of epithelial wound healing [155]. EGF immunoreactivity is low in epithelial cells and bronchial glands of non-asthmatic airways but strong on bronchial epithelium, glands, and smooth muscle in asthmatic airways[156]. Furthermore, in non-asthmatic airways EGF receptor expression is restricted to the bronchial epithelium and glands while in asthmatic airways strong staining is extended to the bronchial smooth muscle and basement membrane [156]. Expression of the EGF receptor reflects the level of epithelial injury in asthma and is proportional to asthma severity [152, 155]. Enomoto et al [157] show sputum levels of EGF to be high during and throughout the recovery phase of an acute attack compared to stable asthmatic patients and control patients. In murine OVA models, treatment of mice with a EGF receptor inhibitor decreases inflammation and collagen deposition [158], and in a rat OVA model an EGF receptor inhibitor abrogates ASM cell growth and goblet cell hyperplasia [159] suggesting EGF receptor inhibition may be beneficial to asthma pathogenesis. There is no data regarding transcriptional regulation of human EGF and only a single paper describing some basic features of the murine EGF promoter, including the transcription start site and consensus binding sequences for NFB, gamma activated sites (GAS), AP-1, AP-2, AP-3, Sp1, p53, hormone response element (HRE) and C/EBP [160]. Thus the transcriptional regulation of EGF remains mostly unexplored. Data regarding the affect of 2 adreoceptor agonists and glucocorticoids on EGF expression is also limited. Glucocorticoids can increase EGF receptor binding via a mechanism at least partly dependent on synthesis of new EGF receptor protein and putatively via glucocorticoid receptor dependent transcription, while 2adreonoceptor agonists reduce EGF receptor binding by an undetermined mechanism [161].


4.3. Insulin-like Growth Factors (IGF) Insulin-like growth factors I and II are single chain polypeptides with high homology to insulin. Both signal via the IGF1receptor tyrosine kinase to activate signalling pathways that favour cell proliferation and survival [162]. The IGF2-receptor preferentially binds IGF-II but has no kinase domain and may not signal [163, 164]. IGF-1 is secreted from mechanically damaged epithelial cells and its inhibition prevents epithelial damage induced fibroblast proliferation [165]. Rabbit ASM cells have IGF-1 and IGF-2 receptors and secrete IGF-II. Furthermore, IGF-I and IGF-II stimulation of rabbit ASM cells induces proliferation via IGF-1 receptor binding. IGF-I also induces human ASM cell proliferation [166] and contraction [167] while IGF-II induces lung fibroblast proliferation [162]. Ovalbumin (OVA) inhalation in a murine model allows detection IGF-I protein in serum and bronchoalveolar lavage fluid (BALF), staining for IGF-I in macrophages and airway epithelial cells and whole lung mRNA expression of IGF-I [164]. Additionally anti-IGF-I antibody infusion inhibits OVA induced airway wall thickening, subepithelial fibrosis and inflammation resulting in reduced airway responsiveness to acetylcholine [164]. The IGF-I gene includes 6 exons and is under the control of two promoters which precede exons 1 and 2 and regulate IGF-Is differential tissue expression [168]. The initial transcripts undergo different RNA splicing and polyadenylation to create multiple mature mRNA species which encode distinct IGF-I precursor proteins that are eventually secreted and processed into mature IGF-I [169] . The best characterised transcriptional response of IGF-I is to growth hormone in the liver. This requires regulation of transcription factor binding and chromatin structure. Growth hormone induces acetylation of both histone H3 and H4 at promoter 1 and 2 [168] partially due to the action of the HAT, p300. It also increases the trimethylation of histone H3 lysine4 (H3K4). Trimethylation of H3K4 is characteristic of actively transcribing genes. Increased histone acetylation and trimethylation is accompanied by activation of RNA polymerase II. Stat5, a member of the signal transduction and activator of transcription (Stat) family, signals downstream of growth hormone and mice lacking Stat5a and Stat5b have reduced expression levels of IGF-I implicating Stats in the transcriptional regulation of IGF-I in response to growth hormone [170]. Indeed IGF-I transcription from both promoters is inhibited by expression of dominant negative Stat5b and induced by expression of constitutively active Stat5b in rats [170] and two adjacent Stat5 binding sites (5’-TTCNNNGAA3’ (where N is G, A,T, or C) exist in the second intron of the rat IGF-I gene, to which Stat5b binds prior to initiation of transcription from the IGF-I promoters [171]. These Stat5 binding sites are thought of as an intronic enhancer and referred to as HS7. More recently two Stat5 binding sites were identified ~77kb 5’ to exon 1 of the rat IGF-I gene that was also preserved in the human gene [169]. Very recently, Chia et al [172] characterised seven Stat5b binding domains that can integrate growth hormone induced IGF-I expression, four of which display potential epigenetic signatures of transcriptional enhancers i.e. binding of the p300, the transcriptional coactivator MED1 and RNA polymerase II. Other transcriptional regulators of IGF-I include: • the SV40 large T antigen-p53 complex in SV40-transformed human mesothelial cells (S-HML), via large T antigen-p53 complex binding to the IGF-I promoter in association with p300 and pRb, in which p300 binding is critically required [173] • hypoxia in HepG2 cells via Stat5b upregulation and binding to the 5’ Stat5 binding sites [174] • testosterone in HepG2 and LNCaP (androgen-sensitive human prostate adenocarcinoma) cells via an androgen responsive region in the IGF-I promoter between -1320 and -1420 bases up-

Clifford et al.

stream of the first codon. This region contains two putative androgen receptor binding sites which bind the androgen receptor in response to testosterone [175] The IGF-II gene spans 30kb and includes 10 exons. It is under the control of 4 promoters (P1-P4) which precede exons 1, 4, 6 and 7 and result in the transcription of 6 different mRNA species [176]. Exons 8 to 10 encode the mature protein, thus each transcript generates the same IGF-II peptide [162]. However the four promoters (under control of different transcription factors), and six transcripts (with varying 5’ untranslated regions, translatability and stability), regulate the spatial, temporal and level of protein expression [177]. IGF-II is induced by hypoxia in HepG2 human hepatoma cells via its P3 promoter, upregulation of the transcription factor Egr-1 and enhanced binding of Egr-1 to the IGF-II promoter [176]. Over expression of IGF-II in fibroblasts from patients with Systemic Sclerosis-Associated Pulmonary Fibrosis also relies on P3 dependent transcription [162]. DNA methylation is a mechanism to silence gene transcription. Variation in methylation status of IGF-IIs four promoter regions occurs in ovarian cancer and differential promoter methylation is associated with disease characteristics and prognosis [177]. In addition aberrant P4 hypomethylation and a correlative increase in IGF-II expression is present in human hepatocarcinoma tissue [178]. The corticosteroid beclomethasone dipropionate, given twice daily to asthmatic patients reduces IGF-I expression in bronchial biopsy samples, and IGF-I concentrations positively correlate with collagen thickening and fibroblast number in the samples [179]. No full mechanism of corticosteroid effect on IGF-I expression has been determined. In osteoblast-enriched cells derived from fetal rat calvaria (Ob cells) cortisol inhibits IGF-I expression via a transcriptional mechanism requiring a glucocorticoid-responsive region of the rat IGF-I exon 1 promoter that is localized to 34 to 192 bp relative to the start site of transcription [180]. 4.4. Vascular Endothelial Growth Factor A (VEGF-A) VEGF-A (from here referred to as VEGF) was identified as an endothelial cell growth factor and vascular permeability factor. It functions as a mitogen, survival and differentiation factor for endothelial cells [181]. It is a factor critical to angiogenesis. Bronchial biopsies from mild asthmatics are more vascular than those from controls and asthmatic bronchial vessels are larger than those of control subjects [182, 183]. Increased vessel number extends into the medium and small airways and vascularity is increased in moderate asthmatics compared to mild asthmatics [184]. Additionally, in asthmatics the number of vessels in the medium airways and FEV1 percentage of predicted are inversely correlated [184]. VEGF levels in sputum are higher in adult asthmatic patients compared to non-asthmatic patients [185-189] and levels correlate with vessel number [188] suggesting VEGF may contribute to the increased angiogenesis seen in the asthmatic lung. In agreement with this, the VEGF in asthmatic BALF can induce angiogenesis in an in vitro endothelial cell and dermal fibroblast coculture system [190]. Levels of induced sputum VEGF are also higher in asthmatic children during an acute attack compared to control children and the higher the asthma severity the greater the level of VEGF. In addition there is a negative correlation between FEV1 percentage of predicted and VEGF levels in asthmatic children [191]. VEGF staining of airway biopsies also shows higher levels of VEGF in asthmatic airways compared to control [188]. Blood vessels in the asthmatic airway are in a destabilised state and are more permeable than ‘heathly’ vessels [192]. VEGF sputum levels correlate with vascular permeability suggesting a role for VEGF in the increased permeability of asthmatic vessels [185-187]. Investigation of the role of VEGF in murine models has proved difficult as knock out or over expression of VEGF during fetal development causes death [193]. However, development of a lung targeted inducible transgenic system allowed the role of VEGF in


asthma to be investigated further. Over expression of VEGF results in the transgenic mice having denser vasculature consisting of larger vessels than the control mice and increased edema. The transgenic mice also exhibit increased inflammation (B lymphocytes and eosinophils), mucus metaplasia, smooth muscle hyperplasia, collagen deposition, activation of TGF1 and airway hyperresponsiveness [193]. On removal of VEGF elaboration, within 2 weeks BAL and tissue inflammation, dendritic cell numbers, mucus metaplasia, mucin gene expression and angiogenesis return to basal levels but smooth muscle hyperplasia and airway hyper responsiveness are still apparent [193, 194]. Angiogenesis, edema, mucus metaplasia, airway hyperresponsiveness and dendritic cell hyperplasia are nitric oxide dependent [195]. In a murine model of toluene diisocyanaste-induced asthma mice develop airway hyperresponsiveness, airway inflammation and increased VEGF levels and inhibition of VEGF receptors reduced all pathophysiological symptoms [196]. We have shown that ASM cells express and secrete VEGF constitutively and in response to various mediators. VEGF also associates with the extracellular matrix surrounding cultured ASM cells [197]. ASM cell secreted VEGF stimulates ASM cell proliferation [198] and angiogenesis in an in vitro endothelial cell and dermal fibroblast coculture system [199]. Bradykinin induces VEGF via COX-2 induction, PGE2 production, activation of adenyl cyclase and up regulation of cAMP [200]. Interleukin 1 (IL-1) also induces ASM cell secretion of VEGF via a transcriptional mechanism dependent on COX-2, while TGF1 induces VEGF via an unknown transcriptional mechanism [41, 201]. The transcriptional mechanism utilised by PGE2 to increase VEGF production has been characterised. PGE2 binds EP2/EP4 G protein coupled membrane receptors to increase cAMP levels. Increased cAMP levels activate protein kinase A signalling and cause phosphorylation of the transcription factor Sp-1. Sp-1 binds the VEGF promoter at one of three Sp1 binding sites in a region of the promoter between -85 and +50bp realative to the transcription start site, resulting in VEGF transcription [40]. Further stimulators of VEGF from ASM cells include angiotensin II [202], endothelin-1[202], IL-6 in combination soluble IL-6 receptor potentially via STAT3 [203], Oncostatin A via STAT3 (also in synergy with IL-1 via increased mRNA stability) [204], IL-4 (via increased mRNA stability [205]) [206], IL-5 [206], IL-13 [205, 206], TGF1 [41], TGF2, and TGF3 [206], TNF at high concentrations [201] and mechanical strain via phosphoinositide 3kinase, ERK and hypoxia inducible factor 1 [207]. No full induction mechanisms have been identified for these stimuli. ASM cells from asthmatic patients secrete more VEGF than non-asthmatic controls and consequently induce greater angiogenesis in vitro [199] but the mechanism of VEGF hypersecretion has not been determined. Beclomethasone dipropionate reduces VEGF levels in induced sputum but not to control levels [185-187]. Six weeks of inhaled Fluticasone propionate therapy following an acute asthma attack in children reduced VEGF sputum levels but again not to control levels [191]. Three month inhaled fluticasone reduced VEGF staining of bronchial biopsy samples from mild asthmatic patients. The reduced VEGF staining was concomitant and proportional to reduced vessel number and angiogenic sprouting and therefore indicative of down regulation of vascular remodelling. BALF levels of VEGF remained unchanged [208]. Treatment of steroid naïve asthmatic patients with the selective leukotriene receptor antagonist, pranlukast, reduced sputum VEGF levels and airway vascular permeability index [209]. Budesonide inhibits basal, IL-4, Il-5, Il-13, IL-1, TGF1,2, and 3 induced VEGF secretion from ASM cells [206]. Formoterol and salmeterol inhibit TNF induced VEGF from human airway epithelial cells, with formoterol being more potent than salmeterol. No mechanisms of drug effects on VEGF expression have been elucidated [210].


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5. CONCLUSIONS It is clear that severe chronic asthma is characterized by upregulation of a huge number of genes associated with chronic inflammation and remodeling and these genes have complex levels of transcriptional control involving protein kinases, transcription factor activation and promoter binding and chromating remodeling. Some of these processes are targets for current asthma treatments such as -adrenoceptor agonists and glucocorticoids but others are potential targets for novel therapeutic approaches. This area will be one of intense interest over the next 20 years. REFERENCES [1] [2] [3] [4] [5] [6] [7]

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Received: January 14, 2011

Accepted: February 23, 2011

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