Role of Transcription Factors in Inflammatory Lung Diseases

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Thorax 1998;53:601–612 601 Role of transcription factors in inﬂammatory lung diseases Thorax: first published as 10.1136/thx.53.7.601 on 1 July 1998. Downloaded from

Irfan Rahman, William MacNee

Acute and chronic alveolar and/or bronchial nuclear receptor co-activators which facilitate inflammation is thought to be central to the DNA binding of transcription factors,8 notably pathogenesis of many lung disorders such as the CREB-binding protein (CBP), adenovirus asthma, chronic obstructive pulmonary disease E1A-associated protein p300/CBP-associated (COPD), adult respiratory distress syndrome factor (p/CAF), and the steroid receptor (ARDS), and idiopathic pulmonary fibrosis co-activator-1 (SRC-1), which interact with (IPF). The site and specific characteristics of basal transcription factors and transcription the inflammatory responses may be diVerent in machinery and are of particular importance in each of these diseases, but all are characterised the transactivation and regulation of gene tran- by the recruitment to the lungs and activation scription involved in the immune and inflam- of immune and inflammatory cells. These acti- matory responses. Understanding the function vated cells produce cytokines, oxidants and and regulation of basal transcription factors is many other mediators which are involved in fundamental to the study of lung inflammation inflammation.12 Recent data indicate that, in and may provide novel therapeutic strategies addition, airway epithelial cells are able to act for a number of inflammatory lung diseases. In as immune eVector cells by secreting pro- this review we will focus on recent progress in inflammatory mediators, oxidants, and our understanding of the role of the NF-êB, cytokines.3 Once triggered, an inappropriate AP-1, NF-IL6, and NFAT transcription fac- chronic inflammatory response persists in tors in the pathogenesis of inflammatory lung these conditions and is presumed to result in diseases and discuss synergistic interactions lung injury. The intracellular molecular between GR proteins, other families of tran- mechanisms in response to environmental sig- scription factors, and nuclear receptor co- nals, leading to increased gene expression and activators which lead to specific gene regula- biosynthesis of proinflammatory mediators by tion. We will also review the current concepts of pharmacological intervention to modulate airspace inflammatory and epithelial cells, are the activation of these transcription factors. of considerable current interest. It is now recognised that there are gene specific factors http://thorax.bmj.com/ which regulate the transcription of particular NF-êB genes by aYnity binding to specific recognition REGULATION OF NF-êB motifs, which are usually located in the NF-êB was first identified as a nuclear factor upstream (5') promoter region of the gene. that binds the decameric DNA sequence These factors, which are usually located in the 5'-GGGACTTTCC-3' within the intronic cytosol, can translocate to the cell nucleus and, immunoglobulin kappa light chain enhancer in by binding to specific consensus sites, can mature B cells.9 Binding of NF-êB to this DNA upregulate the rate of transcription of the gene sequence is responsible for the inducible activ- on September 30, 2021 by guest. Protected copyright. and therefore increase the formation of mes- ity of the enhancer element present in the senger RNA (mRNA) and the protein of an immunoglobulin gene. NF-êB is a member of inflammatory mediator. Some transcription the Rel family of proteins, a novel family of factors are cell specific, but others are ubiqui- ubiquitous transcription factors sharing a tous. Their activity can be modulated by envi- common structural motif for DNA binding and ronmental signals and they may play a key role dimerisation.10 The Rel family of transcription in immune and inflammatory responses. The factors can be defined as a group of proteins transcription factors about which there is most that share sequence homology over a 300 information in immune and inflammatory amino acid region termed the NF-êB/Rel responses are nuclear factor-kappa (ê)B (NF- domain. These proteins can exist either as êB) and activator protein 1 (AP-1).4 Other homodimers or heterodimers, each with a spe- transcription factors include the nuclear factor cific aYnity for diVerent decamer binding sites for the IL-1 responsive element in the IL-6 fitting the êB motif.10 Several diVerent NF-êB gene (NF-IL6), nuclear factor of activated T proteins have been characterised.10 The classi- cells (NFAT), glucocorticoid receptors (GR), cal activated form of NF-êB is a heterodimer Unit of Respiratory cyclic AMP response element binding proteins which usually consists of two proteins, a 65 kD Medicine, Department (CREB), guanine-adenine and thymine- polypeptide (p65) subunit (also referred to as of Medicine, University of adenine repeats (GATA), E26 transformation Rel A) and a 50 kD polypeptide (p50) subunit. Edinburgh, Royal specific (ets), and the signal transducers and Other subunits, such as p105, a p50 precursor Infirmary, Edinburgh activators of transcription (STAT) families of (NF-êB1), p100, a precursor of p52 (NF- EH3 9YW, UK transcription factors. The binding sites for êB2), c-Rel and Rel B are present in diVerent I Rahman these transcription factors are located in the forms of NF-êB. These subunits form various W MacNee promoter regions of a large variety of genes homodimers and heterodimers which are likely Correspondence to: which are considered to be involved in lung to have diVerent aYnities for DNA and diVer- Professor W MacNee. inflammation.5–7 In addition, there are several ent transactivation potentials of the complexes 602 Rahman, MacNee

Table 1 Activators of NF-êB

Cytokines Thorax: first published as 10.1136/thx.53.7.601 on 1 July 1998. Downloaded from Tumour necrosis factor á and tumour necrosis factor â Interleukin-1â Interleukin-2 Interleukin-6 Interleukin-8 Protein kinase C activators Phorbol esters Platelet activating factor Oxidants Hydrogen peroxide Ozone Hyperoxia Growth factors GM-CSC G-CSF M-CSF Bacteria and viruses Mycobacteria tuberculi Rhinovirus Inﬂuenzavirus Adenovirus Allergens Der p 1 Immunoreceptors T cell receptor MHC class I and II IL-2 receptor Others Lipopolysaccharide Ultraviolet radiation Hypoxia Leukotriene B4 f-Met-Leu-Phe (f-MLP)

ACTIVATORS OF NF-êB NF-êB activation represents the terminal step Figure 1 Model for the mechanism of NF-êB and AP-1 activation. Activation of NF-êB in a signal transduction pathway leading from involves the phosphorylation, ubiquitination, and subsequent proteolytic degradation of the the cell surface to the nucleus. A wide variety of inhibitory protein IêB. Free NF-êB then translocates into the nucleus and binds with its extracellular stimuli trigger the activation of êB consensus sites. Antioxidants such as intracellular glutathione and thioredoxin can NF-êB (table 1) including pro-inflammatory inhibit NF-êB activation. Similarly, AP-1 either c-jun/c-jun (homodimer) or c-fos/c-jun 5 10 (heterodimer) migrates into the nucleus and binds with its TRE consensus regions. cytokines, activators of PKC, allergens such 14 Activation of NF-êB/AP-1 leads to gene expression of inflammatory proteins. as Der p 1, and bacterial and viral http://thorax.bmj.com/ 15–17 required to activate diVerent sets of specific products. It may be through NF-êB activation that rhinovirus and influenza virus can genes. In unstimulated cells NF-êB is found in trigger acute exacerbations of asthma. Experi- the cytoplasm as an inactive non-DNA binding mental infection with rhinovirus activates form, associated with an inhibitor protein NF-êB and stimulates the secretion of inter- called inhibitory êB(IêB) which masks the leukin 6 (IL-6) in nasal epithelial cells.15 nuclear translocation signal and so prevents A conserved cysteine residue has been 10 NF-êB from entering the nucleus (fig 1). shown to be critical for both optimal DNA Upon cell stimulation—for example, by binding and redox regulation of NF-êB on September 30, 2021 by guest. Protected copyright. cytokines—specific kinases phosphorylate the protein.18 Reactive oxygen intermediates IêB-á (IêB kinase complex) and the p105/p65 (ROIs) have been recognised as important complex leading to their ubiquitination (trans- inducers of gene expression via NF-êB and fer of the ubiquitin molecule).11 Ubiquitination AP-1.19 Many of the agents that activate of p105 results in an increase in the rate at NF-êB, such as TNF-á, IL-1â, lipopolysaccha- which p105 is processed to p50, whereas ubiq- ride (LPS), and ultraviolet (UV) irradiation, uitination of IêB-á serves as a signal to the also cause an increase in the cellular produc- proteasome complex (proteolytic enzymes) to tion of ROIs by mitochondria.20 ROIs have degrade rapidly and completely the IêB unit in been directly implicated as second messengers the cytoplasm.12 This critical release of NF-êB in the activation of NF-êB, based upon the (p65/p50) from IêB results in the translocation ability of oxidants to activate NF-êB by oxidation of its cysteine-SH group or by ubiquitina- of active p65/p50 into the nucleus where it 21 22 binds to specific motifs in the promoter regions tion and proteolysis. In addition, hyperoxia, ozone,23 and hydrogen peroxide (H O ),24 iron of target genes. The p50 subunit within the 2 2 catalysed lipid peroxidation,25 or depletion of p50/p65 heterodimer greatly facilitates DNA reduced glutathione (GSH), and subsequent binding, whereas the p65 subunit is required 10 increases in cytosolic oxidized glutathione for adequate transactivation. A p50/p50 (GSSG) in response to oxidative stress rapidly homodimer also binds to êB sites but inhibits, 10 ubiquitinate NF-êB, which is a critical step for rather than triggers, transcription. The im- its activation.26 portance of NF-êB subunits is highlighted by the fact that transgenic animals, which do not INHIBITORY êB (IêB) possess the genes coding for the p50 or Rel B IêB has several isoforms (IêB-á,IêB-â,IêB-ã, components of NF-êB, have defects in both IêB-ä,IêB-å), the most predominant being the immune and inflammatory responses.13 á and â subunits.10 These inhibitors have 5–7 Transcription factors in lung diseases 603

Table 2 Inhibitors of NF-êB radical scavengers pyrrolidine dithiocarbamate (PDTC) and N-acetyl-L-cysteine (NAC), in-

Glucocoticoids hibit NF- B activation by preventing the Thorax: first published as 10.1136/thx.53.7.601 on 1 July 1998. Downloaded from Dexamethasone ê Protease inhibitors inducible decay of IêB-á in response to various N-Tosyl-L-phenylalanine chloromethyl ketone (TPCK) stimuli.35 36 Ná-p-Tosyl-L-lysine chloromethyl ketone (TLCK) Antioxidants Glutathione (GSH) IêB KINASE(S) Thioredoxin During the process of activation of NF-êB sev- N-acetyl-L-cysteine (NAC) Pyrrolidine dithiocarbamate (PDTC) eral signal transduction pathways are involved Vitamin E (fig 2). Recent studies have revealed the Cytokines complexity of this process by demonstrating IL-10 IL-4 direct or indirect involvement of a number of Cyclooxygenase and 5 lipoxygenase inhibitors known kinases in the phosphorylation event(s), ICI230487 particularly NF-êB inducing kinase (NIK) MK866 Tepoxalin which has serine/threonine kinase activity Anti-inflammatory agents homologous to the mitogen activated protein- Aspirin 3-kinase (MAP3K) related kinase.37 However, Sodium salicylate Cyclosporin A a number of other kinases are possibly also Others involved in the activation of NF-êB DNA Gliotoxin Capsaicin binding activity and êB site dependent tran- Curcurmin scription. All of the stimuli which activate cAMP NF-êB act by means of kinases that phosphor- Rapamycin ylate and thus degrade IêB by a proteasome mediated mechanism.11 Recently, a serine/ conserved domains known as ankyrin repeats, threonine kinase, CHUK, downstream of NIK, each consisting of around 30 amino acids, which is an NIK activated IêB-á kinase, has forming a unit which is able to interact with been characterised.38 Similarly, a cytokine NF-êB subunits, thus masking the nuclear responsive IêB kinase (IKK), almost identical localisation signals and preventing activation of to CHUK, has been described which specifi- NF-êB and its translocation to the nucleus.10 cally phosphorylates the critical serines 32 and The IêB-á and IêB-å genes have an êB recog- 36 present on the IêB-á molecule, resulting in nition sequence in their promoter regions.10 27 activation of NF-êB in response to proinflam- Thus, agents such as phorbol esters, which matory cytokines.39 The signalling pathways activate NF-êB, will also induce the synthesis that lead to activation of NF-êB are even more and activation of IêB-á and IkB-å which enters complex because of the large number of diVer-

the nucleus to bind to activated NF-êB and ent signals involved in its activation. It is possi- http://thorax.bmj.com/ causes the return of NF-êB to the cytoplasm, ble that multiple signalling pathways become thereby terminating gene activation.28 The integrated to act on a novel multiprotein IêB importance of the inhibitory eVect of IêBis kinase complex. In the light of this, a large shown by the fact that disruption of IêB-á in active multiprotein complex—the IêB kinase mice results in prolonged activation of NF-êB (IKK)—consisting of IKK-1 (identical to in response to inflammatory stimuli. In addi- CHUK) and IKK-2 has been identified, which tion, animals treated in this way die of contains cytokine inducible IêB kinase activity widespread inflammation.29 that phosphorylates both IêB-á and IêB-â to The IêB family contains other members, form the active heterodimers.40 41 However, on September 30, 2021 by guest. Protected copyright. including Bcl-3, IêB-ã, and IêB-ä.30 The intriguing questions on the composition, regu- relative importance of these diVerent inhibitors lation, and function of the IêB kinase complex in regulating NF-êB activation is not clear, but in response to diverse stimuli remain to be the presence of multiple inhibitors is likely to answered—for example, this complex may not be important in balancing the NF-êB activa- be the unique integrator of the NF-êB tion and specific gene regulation. response so that certain stimuli such as oxidants may follow other pathways to IêB INHIBITORS OF NF-êB phosphorylation and NF-êB activation. Several inhibitors of NF-êB activation have been shown to function by either preventing IêB deg- REGULATION OF NF-êB BY TNF AND IL-1 radation or by inducing IêB-á synthesis (table It is recognised that tumour necrosis factor á 2). The immunosuppressive and anti- (TNF-á) and IL-1â are pro-inflammatory inflammatory actions of glucocorticoids have cytokines involved in the pathogenesis of been shown to be mediated by the induction of various inflammatory diseases. TNF-á and IêB-á synthesis.31 A variety of other compounds IL-1â induce loss of IêB from the cytoplasm, can also inhibit NF-êB activation including which is preceded by IêB phosphorylation and tepoxalin, a dual inhibitor of cyclo-oxygenase polyubiquitination of both p105 and IêB-á.42 and 5-lipoxygenase,32 the anti-inflammatory TNF-á and IL-1â activate NF-êB via distinct cytokine interleukin 10 (IL-10),33 and gliotoxin families of cell surface receptors and protein- derived from Aspergillus fumigatus,34 However, protein interactions (fig 2).38 However, both the mechanism and specificity of inhibition of pathways utilise members of the TNF receptor NF-êB by these agents remains unknown. associated factor (TRAF) family of adaptor Antioxidants, such as the physiological proteins as signal transducers, and both intracellular redox regulators glutathione and pathways converge at the level of the protein thioredoxin, thioredoxin peroxidase, and the kinase NIK.37 TNF dependent trimerisation of 604 Rahman, MacNee Thorax: first published as 10.1136/thx.53.7.601 on 1 July 1998. Downloaded from

Figure 2 Model for the putative pathways for the activation of NF-êB initiated by TNF-á and IL-1. the TNF receptor leads to recruitment to the expression of IêB-á. Thus, newly synthesised cell membrane of adaptor protein which is a IêB-á can feedback inhibit NF-êB. By con- TNF-R associated death domain protein trast, induction of NF-êB by IL-1â or LPS

(TRADD), receptor interacting protein (RIP) results in degradation of both IêB-á and IêB-â, http://thorax.bmj.com/ which is a death domain containing serine/ and the activity of NF-êB persists longer threonine kinase and, subsequently, TRAF2, following stimulation despite the presence of and NIK. This sequence of events results in newly synthesised IêB-á and IêB-â. This is activation of NIK, which in turn activates the because IêB-â is resynthesised as a hypophos- IêB kinase, coupled through phosphorylation phorylated form with an altered conformation of IKK-á and IKK-â in the MAPKK activation that leaves the nuclear localising signal on loop. This activation may occur at the cell NF-êB exposed.45 The newly synthesised membrane or after active NIK is released from IêB-â therefore facilitates transport of NF-êB the receptor complex. IêB-á is then recruited as a stable complex to the nucleus for DNA on September 30, 2021 by guest. Protected copyright. to the activated IêB kinase complex where it is binding in a manner that protects it from phosphorylated by the IKK-á/IKK-â het- cytosolic IêB-á. Thus, the continued expres- erodimer. The phosphorylation of IêB-á leads sion of NF-êB as a result of the hypophospho- to its ubiquitination and degradation by the rylated form of IêB-â may lead to persistent proteasome, culminating in the translocation of expression of the genes induced by IL-1 and active NF-êB into the nucleus. In the case of LPS.44 The mechanism which prevents newly the IL-1 receptor, an interaction between synthesised IêB-á associating with activated TRAF6 and NIK leads to the activation of the NF-êB in the cytosol to inhibit its translocation kinase complex. An alternative signal pathway remains to be elucidated. It may be that the rate that involves a mitogen activated protein of degradation of the two newly synthetised kinase/ERK kinase 1 (MEKK1) or MAPKKK IêBs are diVerent, such that IêB-á is degraded is also possible because MEKK1 is recruited to faster as a more eYcient substrate for the the TNF-á activated IêB complex.43 IKK-á kinase subunit compared with IêB-â.11 Recent studies in the Drosophila homologue of TRANSIENT VERSUS PERSISTENT NF-êB the mammalian IêB-á, a phosphoprotein ACTIVATION cactus, show that it is rapidly phosphorylated Although IêB-â interacts as well with the Rel and degraded subsequent to its biosynthesis in protein dimers as IêB-á, the two proteins response to signaling.46 A further explanation display distinct responses to diVerent NF-êB may be that in immune responses, when inducers. For example, TNF-á or phorbol interferon-gamma (IFN-ã) is present, it may myristate acetate (PMA) cause rapid activation not activate NF-êB itself but may synergisti- of NF-êB and transient and rapid degradation cally enhance TNF-á induced persistent of IêB-á, but not of IêB-â.44 The transient NF-êB activation by a mechanism that involves response is due to the fact that activation of increased IêB-á degradation and de novo deg- NF-êB by these stimuli upregulates the radation of IêB-â.47 Transcription factors in lung diseases 605

Table 3 Inﬂammatory genes regulated by NF-êB the antioxidant NAC downregulates VCAM-1 expression and neutrophil mediated inﬂamma-

Cytokines tion in the lungs.54 This suggests a central role Thorax: first published as 10.1136/thx.53.7.601 on 1 July 1998. Downloaded from Tumour necrosis factor á Interleukin 1â for NF-êB in the activation of genes whose Interleukin 2 products promote the adhesion and extravasa- Interleukin 3 Interleukin 6 tion of leucocytes at sites of inflammation. Interleukin 12 Hypoxia is also known to alter intracellular Growth factors redox state leading to the induction of NF-êB Granulocyte-macrophage colony-stimulating factor 55 (GM-CSF) and AP-1 DNA binding, which is associated Granulocyte colony-stimulating factor (G-CSF) with the induction of ICAM-1, iNOS, and IL-8 Macrophage colony-stimulating factor (M-CSF) gene expression in endothelial cells and Chemokines 56–58 Interleukin 8 monocytes. Redox regulation of NF-êB Macrophage inflammatory protein 1 alpha (MIP-1-alpha) may therefore have a pivotal role in cells of the Macrophage chemotactic protein 1 (MCP-1) immune system by acting as a potent and Gro-á,-â,and-ã Eotaxin pleiotropic transcriptional activator. RANTES Inflammatory mediators ROLE OF NF-êB IN APOPTOSIS Inducible nitric oxide synthase (iNOS) Inducible cyclo-oxygenase 2 Apoptosis is an important process in the reso- 5-lipoxygenase lution of inflammation. A role of NF-êB in this Cytosolic phospholipase A 2 process has also been suggested. TNF- , which C reactive protein á 12-lipoxygenase is a strong inducer of NF-êB, also triggers Adhesion molecules apoptosis via TNF receptor 1.59 However, the Intercellular adhesion molecule 1 (ICAM-1) Vascular cell adhesion molecule 1 (V-CAM-1) pathways leading to apoptosis and NF-êB acti- E-selectin vation may be distinct. By contrast, activation Immunoreceptors of NF-êB has been shown to suppress TNF-á Interleukin-2 receptor (á-chain) 60 T-cell receptor (â-chain) mediated apoptosis. These findings suggest Platelet-activating factor receptor that pro-apoptotic extracellular signals can also CD11b and CD48 induce NF-êB, which in turn probably induces Proto-oncogenes p53, c-myc, ras expression of genes that are anti-apoptotic. It appears that the role of NF-êBinthe regulation of apoptosis diVers from cell to cell ROLE OF NF-êBINPRO-INFLAMMATORY depending on their signalling pathways.61 PROCESSES NF-êB regulates the expression of many genes ACTIVATION OF NF-êB IN INFLAMMATORY LUNG involved in inflammatory responses in the DISEASES 5174849 lungs (table 3). In all inflammatory dis- There is clear evidence for the upregulation of http://thorax.bmj.com/ eases adhesion molecules have a role in the genes for pro-inflammatory mediators in a recruiting inflammatory cells such as neu- number of inflammatory lung diseases. Activa- trophils, eosinophils, and T lymphocytes from tion of NF-êB has been shown in human the circulation to the site of inflammation.50 peripheral blood mononuclear cells, airway NF-êB regulates the expression of several epithelial cells (1HAEù-) and lung tissue in genes that encode adhesion molecules, such as response to proinflammatory cytokines such as intercellular adhesion molecule 1 (ICAM-1), IL-1â, TNF-á, or in response to oxidants.62 63 vascular cell adhesion molecule 1 (VCAM-1), However, there have been relatively few direct and E-selectin. Cytokine induced cell surface measurements of the activation of NF-êBin on September 30, 2021 by guest. Protected copyright. expression of E-selectin, VCAM-1 and inflamed lungs. NF-êB is activated in submu- ICAM-1 and the enhanced secretion of IL-8, cosal cells, in sputum derived macrophages, in monocyte chemoattractant protein 1 (MCP- endothelium, and in epithelial cells in bron- 1), MHC proteins, and other chemokines are chial biopsy specimens from patients with regulated at the transcriptional level in en- asthma.64 It has been reported that in alveolar dothelial cells by the binding of NF-êBtoits macrophages from patients with ARDS NF-êB putative site in the 5' flanking sequences.16 is more activated than in alveolar macrophages In many inflammatory lung diseases such as from critically ill patients with other diseases.65 chronic bronchitis, IPF, ARDS, and human However, experimental evidence linking immunodeficiency virus (HIV) depletion of NF-êB activation to the human lung inflam- intracellular GSH or increased levels of GSSG matory diseases is lacking. are present concomitant with the induction of Several animal models have been developed inflammatory mediators and chemotactic to evaluate the role of NF-êB in lung cytokines.51 52 This suggests that the intracellu- inflammation. Intraperitoneal administration lar redox state of the cell may have a key role in of endotoxin in rats leads to the activation of the regulation of NF-êB and the consequent NF-êB in alveolar macrophages and in lung potentiation of the inflammatory immune tissue which is associated with lung neu- responses in lung cells. As has been mentioned trophilia, epithelial permeability, and lipid earlier, NF-êB is involved in the regulation of peroxidation.54 In a mouse model shock caused genes such as E-selectin, VCAM-1, ICAM-1, by haemorrhage produces rapid activation of IL-8, IL-6, and GM-CSF. These molecules are the NF-êB and the cyclic AMP response downregulated in endothelial cells by treat- element binding protein (CREB) through ment with antioxidants.53 This is supported by xanthine oxidase dependent mechanisms in studies of animal models of endotoxin induced pulmonary mononuclear cells.66 67 It has been NF-êB activation in which administration of suggested that activation of NF-êB occurs 606 Rahman, MacNee

through á-adrenergic stimulation by a mech- radicals leads to increased AP-1 DNA anism involving the generation of reactive oxy- binding75 and release of IL-8.76 Inhibition of gen species (ROS). This may be the mech- AP-1 activation by the antioxidant NAC, as Thorax: first published as 10.1136/thx.53.7.601 on 1 July 1998. Downloaded from anism by which blood loss produces increased well as prevention of the induction of c-fos and cytokine expression in the lungs.68 c-jun mRNA, has also been shown to occur in mesothelial cells following treatment with Activator protein 1 (AP-1) NAC, suggesting a direct interaction of oxi- C-FOS AND C-JUN PROTEINS AND THEIR dants and antioxidants with specific cysteine INTERACTIONS groups which are required for AP-1 binding. Activator protein 1 (AP-1) is an important However, contradictory data have been ob- transcription factor composed of proto- tained concerning the mode of AP-1 regulation oncogenes as heterodimers of c-fos, c-jun or as by reduced thiols, such that NAC enhanced c-jun/c-jun homodimers or activating tran- AP-1 DNA binding aYnity and scription factors (ATF). AP-1 is involved in the transactivation.35 Certain phenolic antioxidants transcriptional control of many inflammatory such as butylated hydroxytoluene and mediators. Activated AP-1 has been shown to disrupt the structure of the nucleosome butylated hydroxyanisole exhibit anti- completely.69 Nucleosome disruption is consid- inflammatory eVects and have been observed ered to be the important first step in the chro- to increase the expression of the c-fos and c-jun matin remodelling process involved in the mRNA substantially and to induce AP-1 DNA 77 initial binding of transcriptional factors to a binding. Clearly the relationship between nucleosomal template which allows the tran- oxidants/antioxidants and transcription factor scriptional machinery to function. Dimerisa- regulation is complex and requires further tion of fos-jun or jun-jun is a prerequisite for study. DNA binding via a “leucine zipper” domain which serves as a crucial regulator of the ROLE OF AP-1 IN INFLAMMATORY PROCESSES expression of a wide variety of genes.70 AP-1 activation is essential for cellular prolif- However, such interaction is limited to the het- eration and diVerentiation.70 However, data erodimers of c-fos and c-jun only. Other members of the fos and jun family of proteins are suggesting a role for AP-1 in inflammatory also known to interact with each other but are responses are few. Recent data indicating that unable to activate AP-1 mediated transcrip- redox sensitive JNK and p38 are activated by tion. All of the Jun family of proteins are capa- pro-inflammatory cytokines, such as TNF-á ble of forming homodimers and heterodimers and IL-1, suggest that these signalling path- which can bind to a cognate DNA site. Fos ways may also have a role in inflammatory

proteins do not associate with each other, but responses. Pharmacological inhibitors of p38 http://thorax.bmj.com/ are capable of associating with any members of block many inflammatory responses, especially the Jun family to form stable dimers that have the production of cytokines IL-1, TNF-á, and higher DNA binding activity than Jun dimers. IL-6.78 The promoters of many inflammatory response genes, especially those encoding REGULATION OF AP-1 cytokines and chemokines, have AP-1 binding Activation of the tumour promoter 12-O- sites. This suggests a possible role for JNK in tetradecanoylphorbol-13-acetate (TPA) re- their regulation. JNK is also thought to be sponse element (TRE; this element has the involved in the induction of cyclo-oxygenase base sequence TGACTCA) is a prerequisite 2/prostaglandin synthase 2 which plays an on September 30, 2021 by guest. Protected copyright. for AP-1 induced transcriptional regulation. important part in the inflammatory response Hence, any agents which activate TRE, such as by catalysing the production of serum responsive factor, also activate AP-1 by prostaglandins.79 Platelet activating factor a protein kinase C mediated pathway. Evidence (PAF) has been implicated in the pathogenesis suggests direct involvement of ROS in AP-1 of allergic and inflammatory events in the activation by the c-jun N-terminal kinases airways. Binding of PAF to its receptor on (JNKs), which are members of the MAP kinase 71 human bronchial epithelial cells leads to superfamily. TNF-á induced ROS produc- activation of AP-1-mediated transcription.80 tion acts as a common signal to stimulate AP-1 Direct measurements of components of activation and gene expression by a novel stress AP-1 in disease processes are lacking. AP-1 activated protein kinase (SAPK)/JNK.72 Mi- and its components c-fos and c-jun are overex- togen induced AP-1 activation has been shown 81 to be antioxidant inhibitable, suggesting that it pressed in lungs of smokers. Expression of is ROS mediated.73 c-jun and c-fos were induced in alveolar However, little is known of the exact macrophages and type II pneumocytes in a rat mechanism underlying ROS dependent AP-1 model of lung fibrosis after intratracheal 82 activation. Perturbation of cellular thiol redox administration of bleomycin. Interestingly, status has been suggested as a signal that may c-fos is induced in epithelial cells in asthmatic be involved in the induction of c-fos and c-jun subjects, suggesting a role for AP-1 in the expression, since the intracellular GSH/GSSG regulation of the inflammation in asthma.83 ratio is a key regulator for the induction of More work will be needed to determine stress activated signal transduction pathways whether and how the JNK and p38 pathways by JNK and p38 kinase.74 Depletion of can mediate inflammatory responses and how intracellular GSH in airway epithelial cells fol- the components of AP-1 are involved in the lowing exposure to cigarette smoke free pathogenesis of lung diseases. Transcription factors in lung diseases 607

Nuclear factor-interleukin 6 (NF-IL6) phosphorylation of NFAT proteins and their NF-IL6 was originally identiﬁed as a DNA translocation to the nucleus where they have

binding protein responsible for IL-1 stimulated strong binding aYnity to DNA. The amplitude Thorax: first published as 10.1136/thx.53.7.601 on 1 July 1998. Downloaded from IL-6 induction.84 It has a sequence ACATT- and duration of calcium signals in B cells has GACAATCT and direct cloning of NF-IL6 been shown to control the diVerential action of has revealed that it recognises the same nucle- NF-êB and NFAT. NF-êB and JNK are selec- otide sequences as CCAAT enhancer binding tively activated by a large transient rise in protein (C/EBP).7 NF-IL6 is rapidly phospho- intracellular calcium whereas NFAT is acti- rylated and synergistically induced by LPS or vated by a low sustained rise in calcium.90 inflammatory cytokines such as IL-1, TNF, and IL-6. NF-IL6 can also bind to the regula- ROLE OF NFAT IN IMMUNE INFLAMMATORY tory region of various genes including IL-8, PROCESSES G-CSF, IL-1, and immunoglobulin genes.85 Stimulated T cells transcribe a large array of Furthermore, NF-IL6 has been shown to be activation associated genes, many of which are identical to IL-6DBP, a DNA binding protein potential targets for NFAT; these genes encode responsible for IL-6 mediated induction of transcription factors, signalling proteins, cy- acute phase proteins, demonstrating that NF- tokines IL-2, IL-4, IL-5, IL-8, GM-CSF, IL6 is involved in the expression of the genes TNF-á, cell surface receptors, and other eVec- regulated by IL-6.7 Like NF-êB, NF-IL6 is also tor proteins.6 NFAT plays an important role in considered to be a pleiotropic mediator of regulating the T helper 1 and 2 (Th1 and Th2) many inducible genes involved in acute, patterns of cytokine production. Th1 cells pro- immune, and inflammatory responses, such as duce IL-2 and IFN-ã whereas Th2 cells the genes for IL-7, IL-8, ICAM-1, iNOS, produce IL-4, IL-5, IL-1, and IL-13. Thus, cyclo-oxygenase-2 genes, and several acute NFAT influences T lymphocyte activation and phase protein genes.7 the cytokine profiles of T cells by diVerentiat- ing Th1 and Th2 pathways, which may be ROLE OF NF-IL6 IN INFLAMMATORY PROCESSES important in the pathogenesis of many immune In normal lung tissue NF-IL6 mRNA and disorders. Putative GATA-3, together with NF-IL6 protein are almost undetectable.86 NFAT sites, are present on the promoter of However, infection of human type II pulmo- IL-5 which may be responsible for the nary alveolar epithelial cells with respiratory induction of Th2 type cytokines.91 Since IL-5 is syncytial virus (RSV) leads to NF-IL6 gene produced by CD4+ Th 2 cells, but not by Th1 expression and its synthesis.86 NF-IL6 is cells, NFAT may play a key role in the develop- predominantly expressed in macrophages and ment of eosinophilia in asthma. NFAT proteins is activated in response to IL-1, TNF-á, and show a characteristic ability to interact with

LPS. NF-IL6 regulates the E1A of adenovirus AP-1 and NF-êB DNA binding and transacti- http://thorax.bmj.com/ responsive promoters.87 Adenovirus E1A pro- vation. It has been shown that coupled teins modulate ICAM-1 induction by LPS in NFAT:AP-1 is more stable and has higher airway epithelial cells and may amplify the aYnity for DNA.92 Such interactions are inflammatory processes in the airways of important for the regulation of IL-2, IL-4, smokers and patients with COPD.88 IL-6 levels IL-5, CD40L, GM-CSF gene expression.6 have been shown to be higher in bronchoalveo- As substrates for calcineurin, NFAT proteins lar lavage fluid from patients with lung fibrosis are major targets of the immunosuppressive and those with work related histories of long drugs cyclosporin A and FK506 because of term asbestos exposure.89 In vitro studies on their ability to inhibit dephosphorylation of on September 30, 2021 by guest. Protected copyright. the eVect of asbestos fibres on an airway NFAT.93 An understanding of the regulation epithelial cell line showed induction of IL-6, and function of NFAT proteins may provide us which is associated with the activation of both with selective inhibitors of individual NFAT NF-IL6 and NF-êB, which may result from an proteins in specific cell types or for selected oxidant induced mechanism.89 inducible genes as a means of modulating immune responses. Nuclear factor of activated T cells (NFAT) The nuclear factor of activated T cells (NFAT) Intracellular interactions of transcription factors was first identified in the IL-2 promoter in involved in pro-inflammatory processes human T cells. Transcription factors of the The expression of most genes is controlled by NFAT family regulate the production of eVec- the concerted action of multiple distinct tor proteins that coordinate the immune transcription factors. For instance, the pro- response. Despite their name, NFAT proteins moter of the IL-2 gene contains DNA binding are expressed not only in T cells, but also in sites for at least four distinct transcription fac- other classes of immune system cells ranging tors: NF-êB, AP-1, NFAT, and Oct factors.94 from mast cells, monocytes, macrophages, and Some of these factors can physically interact eosinophils to endothelial cells.6 NFAT pro- with each other when bound to DNA and teins are activated by stimulation of receptors thereby synergistically promote DNA binding coupled to calcium mobilisation. Receptor and transactivation. For example, NF-êB has stimulation and calcium mobilisation result in been shown to interact directly with AP-1 activation of many intracellular enzymes, in- subunits95 and with NFAT within the IFN-ã cluding the calcium and calmodulin dependent promoter,96 whereas AP-1 coordinately inter- phosphatase calcineurin, a major upstream acts with the ets transcription factor in the col- regulator of NFAT proteins. Stimuli that elicit lagenase promoter.97 NFAT also binds AP-1 calcium mobilisation result in the rapid de- and recruits this factor to DNA.6 NF-êB is also 608 Rahman, MacNee

known to synergise with a number of diVerent the activation of NF-êB and AP-1, which may transcriptional activator proteins, including account for most of their anti-inﬂammatory

Sp1, ets, c-Jun, and NF-IL6. This synergy is a actions. However, the relative importance of Thorax: first published as 10.1136/thx.53.7.601 on 1 July 1998. Downloaded from consequence of the direct interactions between these diVerent mechanisms to the NF-êB and these proteins and cooperative immunosuppressive/anti-inflammatory actions binding to adjacent binding sites. The interac- of glucocorticoids remains to be established. tive transcription factor can be present consti- Some in vivo support for this hypothesis comes tutively or co-induced with NF-êB. An exam- from studies in mild asthmatic subjects in ple of the latter is the recent finding that several whom treatment with corticosteroids sup- inducers of NF-êB activate the c-Jun pressed the activation of NF-êB and compo- N-terminal protein kinase (JNK)/p38 which, in nents of AP-1 in the bronchial mucosa turn, phosphorylates c-Jun and synergises with obtained by bronchial biopsy.83 NF-êB.98 Upregulation of E-selectin gene Since glucocorticoids inhibit both NF-êB expression by TNF-á and induction of IL-8 and AP-1 activities, although by diVerent and iNOS gene may also involve a similar mechanisms, it is not surprising that they mechanism.99 100 On the other hand IL-4, decrease expression of a very wide range of which is an immunoregulatory cytokine se- immunoregulatory genes. It is also likely that creted from activated Th2 lymphocytes, eosi- they regulate apoptosis by direct physical inter- nophils, and mast cells, activates the transcrip- actions with p65/AP-1. However, glucocorti- tion factor STAT6101 which competes for coids have opposite eVects on apoptosis in binding to NF-êB binding sites. This competi- neutrophils and eosinophils.108 tion leads to suppression of TNF-á induced E-selectin gene expression in human vascular endothelial cells.101 Thus, physical interactions between transcription factors modulate the Nuclear receptor co-activators expression of numerous inflammatory prod- Steroid receptors and co-activator proteins are ucts such as adhesion molecules, cytokines, known to stimulate gene expression by facilitat- chemokines and growth factors, which are ing the assembly of basal transcription factors involved in the inflammatory responses in into a stable pre-initiation complex. Thus, the lungs. transcription factors gain access to transcrip- tionally repressed chromatin to modulate the transactivation of specific gene networks. Anti-inflammatory eVect of Acetylation of chromatin in vivo is coupled to glucocorticoids on transcription factors Glucocorticoids have been used for decades to transcription. Specific histone acetyltrans- suppress both the immune response and ferases (HATs) target histones bound to DNA and overcome the inhibitory eVect of chroma- inflammation, yet the immunosuppressive http://thorax.bmj.com/ mechanism by which these drugs act is poorly tin on gene expression. High levels of histone understood. There is increasing evidence that acetylation and linker histone deficiency are correlated with gene activity and reduced levels glucocorticoids inhibit the action of transcrip- 109 102 with gene silencing. A few proteins have been tion factors such as AP-1 and NF-êB. Glucocorticoids increase the transcription of identified as nuclear HATs; these include steroid receptor co-activator 1,110 p300/CBP asso- the gene encoding IêB-á and thus suppress the ciated factor (P/CAF),111 p300/CREB-binding inflammatory process by inhibition of IL-2, 112 113 IL-6, IL-8, E-selectin, and iNOS gene protein (CBP), and TAFII230/250. expression.103 104 In the cytoplasm glucocorti- on September 30, 2021 by guest. Protected copyright. coids activate an intracellular receptor protein, CREB-BINDING PROTEIN (CBP ) AND P300 the glucocorticoid receptor. Binding of the glu- CBP and adenovirus E1A associated protein cocorticoid to its receptor transduces the p300 play essential co-activator roles for a hormone signal to the nucleus as a transcrip- number of transcription factors including tion factor.102 Here it binds as a homodimer to CREB, AP-1, GR, and STAT.8 CBP and p300 glucocorticoid response elements (GRE) in could interact simultaneously with more than steroid responsive target genes, resulting in one class of transcription factor. The amounts either induction or repression of gene tran- of these co-activator proteins are tightly scription. However, glucocorticoids decrease controlled. Thus, for promoters that are the transcription of the genes involved in regulated by CBP dependent transcription fac- inflammation which have no identifiable gluco- tors, rates of transcription will depend not only corticoid response elements in their promoter on the levels and activation state of bound regions, suggesting that some other mechanism transcription factors, but also on the cellular must mediate some of the inhibitory eVects of levels of CBP/p300 and the extent to which these hormones. One explanation is that there these co-activators are utilised elsewhere. For may be a direct protein-protein coupling/ example, the glucocorticoid receptor is the interaction between the glucocorticoid recep- most eYcient inhibitor of AP-1 activity.105 tor and AP-1105 and between the receptor and Recent studies have suggested that CBP and NF-êB.106 Thus, the activated glucocorticoid p300 also function as essential co-activators for receptors may then bind to activated NF-êB, several nuclear receptors including glucocorti- AP-1, or CREB and prevent these transcrip- coid receptors.110 Hence, glucocorticoid recep- tion factors from binding to their respective tor antagonism of AP-1 activation may occur consensus regions on genes. These interactions by competition of glucocorticoid receptors may occur in the cytoplasm or the nucleus.107 with these co-activators and therefore accounts Thus, glucocorticoids are potent inhibitors of for transrepression of genes.114 Transcription factors in lung diseases 609

STEROID RECEPTOR CO-ACTIVATOR 1 (SRC-1) inﬂammatory disease states should provide a SRC-1 is a co-activator for many members of molecular mechanism for a novel approach to

the steroid hormone receptor superfamily of therapy. Thorax: first published as 10.1136/thx.53.7.601 on 1 July 1998. Downloaded from ligand inducible transcription factors. SRC-1 possesses intrinsic HAT activity. It also inter- Therapeutic implications acts with another HAT, p300/CBP associated As described above, NF-êB and AP-1 are acti- factor (P/CAF).110 vated by many of the factors which enhance the It is likely that these co-activators and inflammatory response. This activation in turn P/CAF are recruited to the promoter regions of leads to the coordinated expression of many steroid responsive genes in vivo by interaction genes that encode proteins involved in media- with steroid receptors that bind specific DNA tor synthesis and the further amplification and response elements in response to ligand. The perpetuation of the inflammatory response. basal levels of transcription of most genes NF-êB and AP-1 are therefore obvious targets appear to be maintained by histone deacetyla- for novel anti-inflammatory treatments. Gluco- tion which limits access to transcription factors corticoids are eVective inhibitors of NF-êB and and represses gene activation.115 However, AP-1 but have endocrine and metabolic side intrinsic acetyltransferase activity of co- eVects when given systemically. More specific activators such as SRC-1, CBP and P/CAF NF-êB and AP-1 inhibitors could be devel- may alter the existing equilibrium between his- oped which may have less serious side eVects. tone acetylation and histone deacetylation Currently there is much interest in identifying towards the progressive accumulation of nu- more specific and eVective NF-êB inhibitors.20 cleosomes containing acetylated histones.115 These include specific targets in the signalling This targeted histone acetylation may contrib- pathway such as cytokine receptors, the DNA- ute directly to the transcriptional activation binding NF-êB dimer itself, ubiquitin conju- process by disrupting the repressive chromatin gating enzymes, and the proteasome. The most structure and allowing formation of the pre- obvious and specific choice would be a initiation complex in the region of the pro- cytokine responsive IêB multiprotein kinase moter nucleosome which contains the TATA complex (IKK)-IêB-á and IêB-â kinases which box and/or initiator element. It is, however, have been recently shown to activate the IêBin unclear how histone acetylation and deacetyla- response to TNF and IL-1.117 It is probable tion in vivo aVect steroid receptor transduction that screening for specific IKK inhibitors, in of gene expression. particular, which modulate the activation and function of the IKK (IKK-1/2) is likely to have therapeutic value in the modulation of inflam- Implications of glucocorticoid receptor matory responses. In addition, identification of

binding with nuclear co-activators in the other components of the IêB kinase http://thorax.bmj.com/ inflammatory processes complex will allow this to be targeted with In many chronic inflammatory disorders, such novel drugs. It may also be possible to target as asthma, glucocorticoid insensitivity is a rare various IêBs which regulate subsets of NF-êB but challenging clinical problem. The molecu- responsive genes. A better understanding of lar basis of glucocorticoid insensitivity, how- JNK and p38 pathways in the mechanism of ever, is unknown. Abnormal numbers and activation of AP-1 is required for the develop- availability or recruitment of nuclear receptor ment of more specific anti-inflammatory drugs co-activators, their antagonism with other basal against the genes regulated by AP-1. transcription factors such as AP-1, or their Biochemical and clinical studies indicate on September 30, 2021 by guest. Protected copyright. intrinsic acetyltransferase/deacetylase activity that antioxidant therapy may be useful in the and chromatin remodelling, may have an treatment of a wide variety of diseases by opti- important role in controlling the rate of mising the redox and antioxidant defences, or transcription/repression of genes in various by inhibiting NF-êB activation.118 Activation inflammatory lung diseases. In patients whose and binding of transcription factors to consen- asthma is resistant to the anti-inflammatory sus sites on DNA may be driven by the physi- eVects of glucocorticoids, there appears to be ological redox homeostasis, especially by the

exaggerated activation of AP-1. NF-êB and â2 intracellular thiol-disulphide balance. The en- agonists activate CREB that binds to and dogenous glutathione and thioredoxin systems therefore sequesters activated glucocorticoid may therefore be target molecules to modulate receptors inside the nucleus.116 This will reduce the expression of redox sensitive cytokine, the availability of glucocorticoid receptors to interleukin, and adhesion molecule gene inhibit NF-êB or AP-1 or CREB which are expression.118 normally active in such patients. It is also pos- It may not, however, be logical to block the sible that these abnormal glucocorticoid recep- activation of NF-êB for prolonged periods since tors will not repress the transcription machin- it plays such a critical part in the immune and ery due to alteration in HAT activity of other defence responses. Targeted disruption co-activators which have impaired binding to (or knockout) of the p65 component of NF-êB the glucocorticoid response element. The pre- is lethal in animal models because of associated cise role of glucocorticoids on various tran- developmental abnormalities119 whereas the scription factors and co-activators in the regu- lack of the p50 component results in immune lation of various cytokines, particularly in the deﬁciency and increased susceptibility to inﬂammatory process, is an intense area of infection.13 Since NF-êB often works in concert investigation. Understanding the mechanisms with other transcription factors, it may be pos- of the transcriptional machinery in normal and sible to achieve a more selective blockade in 610 Rahman, MacNee

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