Inhibitors of p38 Mitogen-Activated Protein Kinase

Inhibitors of p38 Mitogen-Activated Protein KinasePotential as Anti-inflammatory Agents in Asthma?

Robert Newton1,2 and Neil Holden1

1 Department of Biological Sciences, University of Warwick, Coventry, UK2 Thoracic Medicine, National Heart & Lung Institute, Imperial College Faculty of Medicine, London, UK

Contents Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1131. Asthma and Current Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1142. p38 Mitogen-Activated Protein Kinase (MAPK) as a Potential Therapeutic Target . . . . . . . . . . . . . . . . . . . . . . . . . . . 1143. MAPK Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1144. The p38 MAPK Cascade . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

4.1 p38 MAPK and its Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1154.2 MAPK/extracellular regulated kinase kinase (MEK) and Upstream Activation of p38 MAPK . . . . . . . . . . . . . . . . . 1164.3 Downstream Effectors of the p38 MAPK Cascade . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116

5. Effect of the p38 MAPK Cascade on Gene Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1175.1 Transcriptional Effects of the p38 MAPK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1175.2 Posttranscriptional and Translational Events Are Regulated by p38 MAPK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1185.3 p38-Dependent Responses May Also Be Inhibitory! . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

6. The p38 MAPK in Inflammation and Asthma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1206.1 p38 MAPK in Eosinophils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1206.2 p38 MAPK in Airway Epithelial and Goblet Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1216.3 p38 MAPK in Airway Smooth Muscle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1216.4 p38 MAPK in Endothelial Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1226.5 p38 MAPK in Mast Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1226.6 p38 MAPK in Lymphocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1226.7 p38 MAPK in Monocyte/Macrophage Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1226.8 p38 MAPK in Neutrophils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1236.9 p38 MAPK in Neurogenic Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1236.10 p38 MAPK in Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

7. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124

Abstract Asthma is an inflammatory disease of the airways, which in patients with mild to moderate symptoms isadequately controlled by either β2-adrenoceptor agonists or corticosteroids, or a combination of both. Despitethis, there are classes of patients that fail to respond to these treatments. In addition, there is a general trendtowards increasing morbidity and mortality due to asthma, which suggests that there is a need for new andimproved treatments. The p38 mitogen-activated protein kinases (MAPKs) represent a point of convergencefor multiple signalling processes that are activated in inflammation and that impact on a diverse range of eventsthat are important in inflammation. Small molecule pyridinyl imidazole inhibitors of p38 MAPK have provedto be highly effective in reducing various parameters of inflammation, in particular cytokine expression. Likecorticosteroids, inhibitors of p38 MAPK appear to be able to repress gene expression at multiple levels, forexample, by transcriptional, posttranscriptional and translational repression, and this raises the possibility of asimilarly broad spectrum of anti-inflammatory activities. Indeed these molecules have proved to be effective innumerous in vitro and in vivo models of inflammation and septicaemia, which suggests that such compoundsmay be effective as therapeutic agents against inflammatory disorders. Despite these very promising indicationsof the possible therapeutic use of p38 MAPK inhibitors, a number of events that are p38-dependent are in fact

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also beneficial to the resolution or modulation of diseases such as asthma. We conclude that the overall effectof p38 MAPK inhibition would be beneficial in inflammatory diseases such as rheumatoid arthritis and asthma.However, these drugs may result in a complex phenotype that will require careful evaluation. Currently, anumber of second or third generation inhibitors of p38 MAPK are being tested in phase I and phase II clinicaltrials.

1. Asthma and Current Therapy

Asthma is a disease of the airways and is characterised byinflammation, hyperreactivity and, frequently, airway remodell-ing (figure 1).[1] Clinically, this is manifested as variable airflowlimitation, shortness of breath (particularly at night), wheeze,chest tightness and often cough. Furthermore, and unlike diseasessuch as chronic obstructive pulmonary disease (COPD), asthmais generally associated with atopy and it is the infiltration of eo-sinophils to the lung that is believed to account for much of thepathophysiology in asthma.[2] In terms of the cost to society, thesocio-economic burden associated with asthma is increasing, asthe prevalence in industrial nations has risen steadily over recentdecades and now approximately 6% of adults in the US are re-ported as having asthma.[3] This prevalence may reach up to 15%in other countries, which, taken with the fact that in the US mor-tality rates due to asthma have increased by 25% since 1960,suggests the need for new and improved treatment regimens.[3-5]

The two mainstay therapies for asthma are currently: (i) shortacting β2-adrenoceptor (β2AR) agonists; and (ii) inhaled cortico-steroids.[6] The majority of mild, medium and even relatively se-vere cases of asthma are adequately controlled by either inhaledβ2AR agonists alone, or a combination of β2AR agonists plusinhaled corticosteroids. Patients with more serious asthma areusually prescribed oral corticosteroids such as prednisolone.From a therapeutic perspective these two classes of drugs areessentially complementary, with β2AR agonists promoting relax-ation of airway’s smooth muscle, targeting airway hyperreactiv-ity and with corticosteroids targeting inflammation (figure 1).[6-8]

However, it is noteworthy that neither class of drug appears toshow any great effect on preexisting airway remodelling.[9,10] Inaddition, there are classes of patient who are resistant to cortico-steroids and are not adequately controlled even by high dose oralcorticosteroids.[11,12] Furthermore, long-term high dose cortico-steroid regimens lead to metabolic, endocrinic and systemic ad-verse effects, whilst other anti-asthma drugs including anticho-linergics, methylxanthines or cromoglycates and more recentlyantileukotrienes are generally of a lesser therapeutic utility. Con-sequently, there remains a genuine need for new anti-asthma ther-apies, particularly those that are able to target airway remodellingin addition to inflammation.[4,5]

2. p38 Mitogen-Activated Protein Kinase (MAPK)as a Potential Therapeutic Target

One class of compounds that show promise as potential ther-apeutic agents in inflammatory diseases is the pyridinyl imida-zoles. These compounds, typified by SKF 86002 (table I) , havebeen documented as showing potent anti-inflammatory propertiesin arachidonic acid-induced rat paw and mouse ear oedema,[13]

rat carrageenan-induced oedema and adjuvant-induced arthri-tis,[14] as well as in mouse collagen-induced arthritis models ofinflammation.[15] In addition, pryridinyl imidazoles potently in-hibit interleukin (IL)-1β release from human monocytes treatedwith lipopolysaccharide (LPS) or IL-1β.[16] Whilst these biolog-ical effects led to the term cytokine-suppressive anti-inflammatorydrug (CSAID), it was not until 1994 with the cloning of the CSAIDbinding protein (CSBP), now known as p38 mitogen-activatedprotein kinase (MAPK) [see table II], that the link between anti-inflammatory effect and drug target was made.[17] We now knowthat this kinase cascade is strongly activated by various pro-inflammatory and stressful stimuli, and initial studies using morepotent p38 MAPK inhibitors have documented a significant re-pressive effect on inflammatory responses in numerous cell-based and in vivo assays (figure 2).[17-20]

3. MAPK Pathways

In common with the MAPK/extracellular regulated kinase(ERK) and stress activated protein kinase (SAPK)/Jun N-terminalkinase (JNK) MAPK cascades, the p38 MAPK is a proline-directed kinase and the p38 signalling cascade consists of a linkedseries of protein kinases that sequentially phosphorylate and ac-tivate the next kinase in the cascade (figure 2) [for review seeKyriakis & Avruch[26]]. In each case, the downstream MAPKscontain a conserved Thr-X-Tyr motif in their activation loops,which is a substrate for MAPK kinases (MAPKKs or MAP2Ks)and is necessary for kinase activation. MAP2Ks, most frequentlyknown as MAPK/ERK kinases (MEKs) or MAPK kinases(MKKs), are themselves acted on by a structurally diverse groupof upstream kinases that are collectively referred to as MAP2Kkinases (MAPKKKs or MAP3Ks) [figure 2]. The activation ofMAP2Ks or MEKs by MAP3Ks requires dual phosphorylation,which unlike the downstream MAPKs takes place on Ser/Thrresidues within the kinase domain. For example, the two MEKs,

114 Newton & Holden

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MKK3 and MKK6, that are responsible for activation of p38MAPK are phosphorylated on Ser-189 and Thr-193, and, Ser-207and Ser-211, respectively[26]. Activation of MAPK cascades, in-cluding p38, may occur following a variety of stimuli including:proinflammatory cytokines, such as tumour necrosis factor-α(TNFα) or IL-1β; growth factors, including epidermal growthfactor (EGF) or platelet-derived growth factor (PDGF); as wellas by a variety of environmental stresses including osmoticshock, ultra violet (UV) irradiation, ionising radiation and isch-aemia or the bacterial cell wall product LPS.[26]

How these stimuli activate MAP3Ks is still equivocal. How-ever, activation of Raf, the MAP3K responsible for activating theMEK-ERK MAPK kinase cascade by the small GTP-bindingprotein, Ras, appears to require recruitment to the plasma mem-brane, oligomerisation and phosphorylation.[26] These complexevents take place within the context of adaptor or scaffold pro-teins, which as well as playing a role in transducing signals fromnearby receptors, are also thought to be important in determiningthe appropriate spatial arrangement, and therefore specificity, tothe various MAPK modules.[26] Thus, in the case of the mamma-lian p38 MAPK cascade, the scaffold protein IB2/JIP2 interactswith upstream Rac-guanine exchange factors (Rac-GEFs), Racand other components of the p38 signalling pathway includingp38 itself.[27]

4. The p38 MAPK Cascade

4.1 p38 MAPK and its Inhibition

In the case of the p38 MAPK cascade the p38 kinase existsas a number of isoforms derived from four homologous genes(table II): p38α,[17,28-31] p38β,[32-34] p38γ[35] and p38δ.[36] Theseall contain the same activation-loop phospho-acceptor site, Thr-Gly-Tyr, but show markedly different sensitivities to thepyridinyl imidazole CSAID class of drugs.[36-38] Thus, the p38αisoform, which was originally characterised on the basis of bind-ing to CSAIDs,[17] is strongly inhibited by pyridinyl imidazolessuch as SB 202190 and SB 203580.[18] Similarly the p38β iso-form, which is most homologous to the p38α isoform, is stronglyinhibited by both SB 202190 and SB 203580, whereas the p38γand p38δ isoforms show a >100 fold lower sensitivity to inhibi-tion by SB 203580. Whilst pyridinyl imidazole inhibitors bindthe ATP binding site and are competitive with respect to ATP,the differences in relative potency are attributed to specific aminoacid differences in p38γ and p38δ at the equivalent position toThr-106 in p38α.[36-39] One important consequence of this aminoacid change is that most biological data have accrued in respectof the p38α and p38β isoforms and it is these isoforms that aretargeted therapeutically.

Asthma

(i) Smooth muscle dysfunction:

• Airway hyperreactivity• Bronchoconstriction

(ii) Airway inflammation:

• Inflammatory cell (especiallyeosinophil) infiltration

• Mucosal oedema• Cytokine release• Mediator release• Expression of

adhesion molecules• Expression of destructive

enzymes (MMPs, etc.)• Expression of inflammatory

enzymes (COX-2, iNOS)• Mucus hypersecretion

(iii) Airway remodelling:

• Smooth muscle proliferation• Epithelial damage• Smooth muscle and

epithelial cell hyperplasia• Basement membrane thickening

Corticosteriodsβ2AR agonists ?

Fig. 1. The tripartite pathology of asthma. The pathophysiology of asthma can be divided broadly into: (i) smooth muscle dysfunction; (ii) airway inflammation; and (iii) airwayremodelling. This schematic shows some of the elements that are associated with each of these phenotypic components and the targeting by standard therapy. The fact that airwayinflammation leads to increased smooth muscle dysfunction and airway remodelling is indicated. β2AR = short acting β2-adrenoceptor; COX-2 = cyclo-oxygenase 2; iNOS = induciblenitric oxide synthase; MMP = matrix metalloproteinase.

p38 MAPK in Asthma 115


4.2 MAPK/extracellular regulated kinase kinase (MEK)and Upstream Activation of p38 MAPK

Upstream of p38 MAPK lie the MEKs, MKK3 (also known asMEK3 or SKK2), which preferentially activates the α and β iso-forms of p38, and MKK6 (also known as MEK6 or SKK3), whichcan potently activate all four p38 MAPKs (table II) [figure 3].[36,37]

These two MEKs demonstrate a relatively high degree of speci-ficity for the p38 MAPKs and are not thought to play a significantrole in activation of either the ERK or SAPK/JNK MAPK path-ways.[36,37,40-42] By contrast MAP3Ks that are capable of phos-phorylating MKK3 and MKK6 are relatively diverse and show asignificant degree of promiscuity with respect to other MAPKsignalling pathways (see Kyriakis & Avruch 2001[26]). Thus, thethousand and one kinases (TAOs), MEK kinases (MEKKs) 2, 3and 4, apoptosis signal-regulating kinase 1 (ASK1), TGF-β acti-vated kinase (TAK1) and the proto-oncogene Tpl-2 may all acti-vate MKK3, whereas MKK6 may only be activated by MEKK2,MEKK3 and MEKK4, ASK1 and TAK1 (figure 3).[26]

This apparent complexity, or possibly lack of clarity, in re-spect of MAP3Ks that activate the p38 MAPKs is also reflectedin the lack of certainty over the upstream mechanisms and pro-teins involved in activating the p38 cascade.[26] As with the ERKpathway, the small GTPase, Ras, may play a role in the IL-1-induced activation of p38, in this case by interacting with theMAP3K, TAK1, and other proteins known to associate with theIL-1 receptor.[43] However, this is generally thought to be a lesserrole for Ras, as in fact Ras-like proteins, including Rac1, Rho andCdc42H, are more strongly implicated in the activation of the p38pathway.[44,45] More recently, there have been suggestions thatheterotrimeric guanine nucleotide-binding proteins (G proteins)can activate p38 MAPK via MKK3 and MKK6. Indeed, the m1muscarinic receptor, a G protein linked receptor that is implicatedin airway hyperresponsiveness, was able to transcriptionally ac-tivate c-Jun.[46,47] This finding, if generally true, raises the possi-

bility of p38 activation by numerous activator molecules actingvia G-protein coupled receptors (GPCRs), and would dramati-cally increase our perception of the role of p38 MAPK in thecontext of complex diseases such as asthma.

Finally, the germinal centre kinases (GCKs) represent agroup of upstream kinases that lie upstream of MAP3Ks and arecapable of activating MAPK pathways, including p38, in over-expression studies.[48-50] However, whilst these kinases may beactivated by proinflammatory and stress stimuli, their true rolesin the activation of MAPK cascades and their bona fide targetsremain to be determined.

4.3 Downstream Effectors of the p38 MAPK Cascade

A number of well-defined downstream targets of the p38MAPK have been identified which include the MAPK-activatedprotein kinases (MAPKAP-Ks or MKs), MAPKAP-K2 andMAPKAP-K3 (figure 3).[51,52] These are both capable of phos-phorylating the small heat shock protein, hsp27, a molecular cha-perone that is believed to participate in both actin rearrangementand stress fibre formation.[52,53] Other kinases that are thought toact downstream of p38 MAPK include: p38-regulated/activatedkinase (PRAK), which is also believed to have hsp27 as a substrate,[54]

mitogen- and stress-activated protein kinases 1 (MSK1) and 2(MSK2)[55,56] and MAPK signal-integrating kinase (MNK1).[57,58]

Whilst the elucidation of downstream effectors for these kinasesis still at an early stage, there is evidence that MSK1, and to alesser extent MSK2, can phosphorylate both cAMP response el-ement binding protein (CREB), at Ser-133, and activating tran-scription factor 1 (ATF1) to transcriptionally activate these tran-scription factors.[55,56] In addition, MNK1 has been shown toregulate mRNA translational via phosphorylation of the transala-tion initiation factor, eIF-4E (figure 3).[58-60]

Another group of pathways that play important physiologicalroles in both inflammation and bronchoconstriction are the

Table I. Inhibitors of p38 Mitogen-Activated Protein Kinase (MAPK)

Common name Chemical name Company Reference

SB 203580 4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl)imidazole GlaxoSmithKline [18]

SB 202190 4-(4-Fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole GlaxoSmithKline [17]

SB 242235 1-(4-piperidinyl)-4-(4-fluorophenyl)-5-(2-methoxy-4-pyrimidinyl)imidazole GlaxoSmithKline [21]

SB 239063 trans-1-(4-hydroxycyclohexyl)-4-(4-fluorophenyl)-5-(2methoxypyridimidin-4-yl)imidazole

GlaxoSmithKline [22]

SKF 86002 6-(4-fluorophenyl)2,3-dihydro-5-(4-pyridinyl)imidazole(2,1-b)thiazole GlaxoSmithKline [14]

L-790070 (S)-5-[2-(1-phenylethylamino)pyrimidin-4-yl]-1-methyl-4-(3-trifluoromethylphenyl)-2-(4-piperidinyl)imidazole

Merck & Co [23]

VX745 5-(2,6-Dichlorophenyl)-2-(phenylthio)-6H-pyrimido[1,6-b]pyridazin-6-one Vertex Pharmaceuticals [24]

RWJ 67657 6-Amino-2-(4-fluorophenyl)-4-methoxy-3-(4-pyridyl)-1H-pyrrolo[2, 3-b]pyridine Johnson & Johnson [25]

116 Newton & Holden


eicosanoid synthetic pathways. Thus 5-lipoxygenase (5LO),which catalyses the first step in the production of the broncho-constrictor leukotrienes from the precursor arachidonic acid, hasshown to be activated at the level of enzyme activity byMAPKAP-K2.[61] In addition, the upstream step, which results inthe release of arachidonic acid from membrane phospholipid, andis catalysed by the cytosolic form of phospholipase A2 (cPLA2),is also p38-dependent and more recently has been shown to in-volve MNK1.[62,63] Thus, inhibition of p38 MAPK may result inbeneficial effects such as the prevention of leukotriene synthesisor reduced synthesis of bronchoconstrictor prostaglandins suchas PGF2α. Alternatively, and depending on the cell type, p38inhibition could reduce synthesis of bronchodilatory prostaglan-dins such as PGE2.[64] In addition, the effect of p38 inhibitors onprostanoid and leukotriene production is confounded by the factthat the initial, although not the more recent, CSAIDs were char-acterised as biochemical inhibitors of 5LO and cyclo-oxygenase(COX), the enzyme responsible for the first dedicated step inprostanoid production.[65]

5. Effect of the p38 MAPK Cascade on Gene Expression

In the above sections, p38 inhibitors, or CSAIDs, were notedas potent inhibitors of cytokine expression. Mechanistically, in-duced cytokine expression, for example, in inflammatory situa-tions, may be regulated transcriptionally, posttranscriptionally,

translationally or posttranslationally. Whilst for any particularcytokine, from a given cell type and in response to a specificstimulus, any one of these mechanisms may predominate, cor-rectly regulated gene expression will generally require a balanceof these different mechanisms. Furthermore, and like the anti-inflammatory actions of corticosteroids,[66] the p38 cascade hasbeen shown to exert effects at various levels of gene regula-tion.[26]

5.1 Transcriptional Effects of the p38 MAPK

The p38 MAPK has been implicated in the activation of var-ious transcription factors including: ATF1,[56,67] ATF2,[68]

CREB,[55,56,67] myocyte enhancer factor 2C (MEF2C),[69] CHOP(GADD153)[70] and Max.[31] The fact that many of these factorsimpact on the proinflammatory transcription factor, activatorprotein-1 (AP-1), and AP-1-like activities, is consistent with arole for p38 activation in the induction of AP-1-dependent tran-scription by the inflammatory cytokine TNFα.[44] Furthermore,AP-1, along with nuclear factor (NF)-κB, an acute phase inflam-matory transcription factor, have been shown to be elevated ininflammatory diseases such as asthma where they may exacer-bate disease pathology by contributing to a positive feed-forwardloop that, unless broken by anti-inflammatory drugs such as cor-ticosteroids, leads to renewed inflammatory gene synthesis andthe perpetuation of inflammation.[66,71,72]

Table II. p38 Mitogen-Activated Protein Kinase (MAPK) cascade nomenclature and substrates

Name Alternatives Level of kinase Common MAPK cascade substrates

TAO1 MAP3K MKK3

TAO2 MAP3K MKK3

MEKK2 MAP3K MEK1/2, SEK1, MKK3, MKK6

MEKK3 MAP3K MEK1/2, SEK1, MKK7, MKK3, MKK6, MEK5

MEKK4 MTK1 MAP3K SEK1, MKK7, MKK3, MKK6

MAP3K8 Tpl-2, Cot MAP3K MKK3

TAK1 MAP3K SEK1, MKK3, MKK6

ASK1 MAP3K5 MAP3K SEK1, MKK7, MKK3, MKK6

MKK3 MEK3, SKK2 MAP2K p38α, p38βMKK6 MEK6, SKK3 MAP2K p38α, p38β, p38γ, p38δp38α SAPK2a, CSBP1, p40, RK, Mxi2 MAPK MAPKAP-K2, MAPKAP-K3, ATF2, MSKs, Elk1,

MEF2C

p38β SAPK2b, p38-2 MAPK MAPKAP-K2, MAPKAP-K3, ATF2, MSKs

p38γ SAPK3, ERK6 MAPK ATF2

p38δ SAPK4 MAPK ATF2

ASK1 = apoptosis signal-regulating kinase 1; ATF = activating transcription factor; ERK = extracellular regulated kinase; MAP2K = mitogen-activated proteinkinase kinase; MAP3K = mitogen-activated protein kinase kinase kinase; MAPKAP-K = MAPK-activated protein kinase; MEF = myocyte enhancer factor;MEK = mitogen-activated kinase kinase; MEKK = mitogen-activated protein kinase/extracellular protein kinase kinase kinase; MKK = MAP kinase kinase;MSK = mitogen- and stress-activated protein kinase; MTK = MAP3 kinase; RK = reactivating kinase; SAPK, SEK = stress-activated protein kinase; SKK = SAPKkinase; TAK = transforming growth factor-β activated kinase; TAO = thousand and one kinase.



Interestingly, transcriptional induction of the AP-1 compo-nents, c-fos and c-Jun, events which require JNK-dependentphosphorylation of ternary complex factor (TCF) and both ATF-2/c-Jun respectively, have been shown to also require p38 MAPKactivity.[73] One target of p38 MAPK that may explain these datais the transcription factor MEF2C, which is phosphorylated inresponse to LPS and acts to elevate c-Jun transcription.[69] Like-wise the MAP3K, Cot/Tpl-2, can activate MKK6 and p38 MAPKand leads to c-Jun expression.[74] Similarly, TCFs, such as Elk-1and Sap-1a, which act via serum response elements (SREs) andare important in immediate early gene expression, including c-fos, are also reported to be p38 MAPK-dependent and will againimpact on AP-1-dependent transcription.[36,37,75,76]

In addition to direct effects on transcription factor activation,a number of studies have implicated the p38 MAPK in the tran-scriptional competency, as distinct from factor activation or DNAbinding, of NF-κB.[44,77-80] Thus inhibition of transcriptional ac-tivity was hypothesised to account for the inhibition of IL-6 syn-thesis by SB 203580.[77] As NF-κB DNA binding and phosphor-ylation of NF-κB subunits were unaffected by inhibitors of p38MAPK, whilst phosphorylation of downstream factors such asTATA-binding protein (TBP) were reduced, it was suggested thatthe p38 MAPK may play a role in the activation of transcriptionby modulating the transcriptional activity of the basal transcrip-tional machinery.[79] Another downstream event that is p38MAPK-dependent and may impact on immediate early inflam-matory gene expression is the phosphorylation of the histone pro-tein H3 and the non-histone structural protein, HMG14.[73,81]

Continuing with this theme, p38 MAPK has now been proposed

to ‘tag’ NF-κB-dependent genes via phosphorylation of histoneH3.[82] In this study, the authors suggest that p38 MAPK activityleads to a change in chromatin structure in the region of certaininflammatory gene promoters and this leads to increased recruit-ment of NF-κB. Given the pivotal role of NF-κB in inflammatorydiseases such as asthma, these data are consistent with a benefi-cial therapeutic effect of p38 MAPK inhibitors.[71] However,other studies have only observed inhibition of NF-κB-dependenttranscription at doses of SB 203580 that are in excess of thosenormally required for inhibition of p38 MAPK.[83] Consequently,a word of caution is necessary as many of the above studies havealso used p38 inhibitors, for example, SB 203580, at similarlyhigh levels.

One further transcriptional effect of p38 MAPK includes themodulation of the signal transducers and activators of transcrip-tion (STAT) proteins. For example, STAT1 and STAT4 tran-scriptional activities were inhibited by SB 202190.[84,85] In thecase of STAT4, full transcriptional activation was shown to in-volve the p38 MAPK and the upstream MEK, MKK6.[85] Con-versely, the inhibition of p38 MAPK by SB 203580 had no effecton interferon (IFN)-γ-dependent STAT1-mediated transcription,but inhibited transcriptional enhancement by LPS.[86] Immuno-logically, STAT1 plays an important role in the development ofT helper type 1 (Th1) type responses by mediating many of theeffects of the Th1 cytokine, IFNγ, whilst STAT4 is important inIL-12 signalling. As IL-12 is necessary for the development ofTh1 phenotypes, and allergic asthma is characterised by a pre-dominance of T cells of the Th2 phenotype over Th1 cells, it ispossible that inhibition of these STAT-dependent functions byp38 MAPK inhibitors may result in a further swing towards a Th2response.

Notwithstanding effects on STATs, the above data representa number of events that are apparently regulated by the p38MAPK and impact, in particular, on AP-1 and NF-κB-dependenttranscription. These findings are of particular relevance in severe/steroid resistant asthma where a resistance of AP-1 to the effectsof corticosteroids is implicated in the disease pathology and theinhibition of p38 MAPK may reestablish corticosteroid respon-siveness.[87,88]

5.2 Posttranscriptional and Translational Events AreRegulated by p38 MAPK

As noted above, the CSAID SKF 86002 inhibited TNFα andIL-1β production from cell lines, macrophage ex vivo, and invivo.[20,89] This effect on cytokine production occurred predomi-nantly at the level of cytokine translation as the reductions incytokine protein were not mirrored at the mRNA level.[90] In the

MAP3K(inactive)

MAP3K

MAP2K

MAPK

MAP3K(active)

MAP2K(active)

MAPK(active)

MAP2K(inactive)

Ser

Ser/Thr

MAPK(inactive)

Thr

Tyr

Ser

Ser/Thr

Thr

Tyr

EffectorsEffectors

Activators Activators (LPS, IL-1, TNFα, UV light, PMA,osmotic shock, growth factors)

Fig. 2. Mitogen-activated protein kinase (MAPK) signal transduction cascades. Astylised MAPK pathway is depicted showing the relationship between MAP3K, MAP2Kand MAPK. On the right hand side, the residues that are typically phosphorylated forkinase activation are shown. IL-1 = interleukin-1; LPS = lipopolysaccharide; PMA =phorbol 12-myristate 13-acetate; TNFα = tumour necrosis factor-α; UV = ultra violet.

118 Newton & Holden


report of Lee et al. 1994,[17,90] the cytokine suppressive pyridinylimidazole derivatives of SKF 86002, including SB 202190 andSB 203580, were shown to be potent inhibitors of the newly dis-covered p38 MAPK, thereby implicating a role for p38 in cyto-kine translation.

In recent years, it has become increasingly obvious that nu-merous genes are controlled at the posttranscriptional and trans-lational level.[91-93] For example, the 3′untranslated region (UTR)of granulocyte-macrophage colony-stimulating factor (GM-CSF)mRNA contains numerous AU-rich elements (AREs) that play arole in mRNA destabilising and translational blockage.[94-96] Thepotential for regulation at this level is illustrated by the fact thatproteins binding to these sites can regulate GM-CSF mRNA turn-over, whilst mutation of AREs enhances expression.[97,98] In ad-dition, AREs have been described in the 3′UTRs of numerousacute phase and inflammatory genes suggesting that similarmechanisms of regulation are in fact widespread.[99] In this re-spect, IL-11 expression in bone marrow stromal cells was highlyinducible by IL-1β and this increase was exclusively attributedto mRNA stabilisation.[100] Likewise, a substantial degree of reg-

ulatory control also occurs at the level of TNFα mRNA transla-tion, and this is thought to involve the binding of specific proteinsto the ARE within the 3′UTR as well as by increases in thepoly(A) length.[101-104]

Whilst posttranscriptional and translational mechanisms havebeen documented in the induction of various inflammatory genesincluding those encoding IL-1, TNFα, IL-2, IL-6, IL-8, GM-CSFand cyclo-oxygenase-2 (COX-2),[104-112] it is now equally clearthat p38 MAPK is a significant player in the control of thesemechanisms. Thus, the p38 MAPK has been implicated in thecontrol of mRNA stability of TNFα,[113,114] IL-3,[115] IL-6, IL-8,GM-CSF,[109] and COX-2,[116,117] and this effect may occur viathe AREs in the 3′UTR of these genes. Similarly, the ability of theprofibrotic cytokine transforming growth factor-β (TGF- β) to up-regulate elastin mRNA is at least in part a p38 MAPK-dependentresponse that involves elastin mRNA stabilisation.[118] In addi-tion, targeted mutation of MAPKAP-K2 resulted in a profoundrepression of TNFα biosynthesis, but not mRNA expression orsecretion.[119] Interestingly, this effect could be restored by dele-tion of the TNFα ARE suggesting that MAPKAP-K2-dependent

MAP3K

MAP2K

MAPK

Effectors

MKK3 MKK6

p38α p38β p38γ p38δ

MAPKAP-K2 MAPKAP-K3 PRAK MSK1/2 MNK1/2 Other targets

ARE-dependentmRNA stability

5LO activity

hsp27 CREBHistone H3

HMG-14ATF1

elF4EcPLA2

CHOPMaxATF2c-JunElk-1

Sap-1aSTAT1 & 4

MEF2CTBP

TAOsTpl-2

MEKK2MEKK3MEKK4TAK1ASK1

Fig. 3. The p38 mitogen-activated protein kinase (MAPK) signal transduction cascade. The four p38 MAPK isoforms are shown along with their two main upstream activators.

Some of the possible upstream activators are also depicted to illustrate the apparent complexity at this level. Downstream of p38 MAPK are the effector kinases and actual effectors

themselves. ARE = AU response element; ASK1 = apoptosis signal-regulating kinase 1; ATF = activating transcription factor; CHOP = CCAAT/enhancer-binding protein (C/EBP)

homologous protein; cPLA2 = cytosolic phospholipase A2; CREB = cyclic AMP response element binding protein; eIF4E = eukaryotic initiation factor 4E; HMG-14 = high mobility

group 14; hsp27 = heat shock protein 27; MAPK = mitogen-activated protein kinase; MAP2K = mitogen-activated protein kinase kinase; MAP3K = mitogen-activated protein kinase

kinase kinase; MAPKAP-K = MAPK-activated protein kinase; MEF2C = myocyte-specific enhancer binding factor 2C; MEKK = mitogen-activated protein kinase/extracellular protein

kinase kinase kinase; MKK = MAP kinase kinase; MNK = MAPK-interacting kinase; mRNA = messenger RNA; MSK = mitogen- and stress-activated protein kinase; PRAK =

p38-related/activated protein kinase; STAT = signal transducer and activator of transcription; TAK = TGFβ activated kinase; TAOs = thousand and one kinases; TBP = TATA

binding protein; 5LO = 5-lipoxygenase.



TNFα biosynthesis involves this element.[120] Furthermore,whilst in these studies TNFα expression was predominantly reg-ulated at the level of translation, this same study found IL-6 to bemainly regulated at the level of mRNA stability suggesting thatthe p38/MAPKAP-K2 pathways can differentially control boththese aspects of gene expression.[120]

Finally, it is encouraging from a therapeutic perspective to notethat the corticosteroid dexamethasone and the anti-inflammatorycytokine, IL-10, are both reported to target the p38 MAPK tocause mRNA destabilisation and translational repression.[117,121]

However, whilst numerous ARE binding proteins have beenidentified, the actual targets of p38-mediated mRNA stabilisationor translational control still remain to be determined.

5.3 p38-Dependent Responses May Also Be Inhibitory!

Rather ironically, one mRNA binding protein that has beenshown to require p38 MAPK for inducible expression istristetraproline (TTP).[107] This protein is thought to play a rolein feed-forward inhibition of immediate early genes, wherebyproinflammatory stimuli activate inflammatory gene expressionand simultaneously induce the p38-dependent expression of TTP.TTP then promotes deadenylation (plus presumably translationalarrest) and destabilisation of mRNA to produce a tightly regu-lated period of gene expression in target genes.[107,122,123] Con-sistent with this model, TTP gene knock-out results in elevatedexpression of the ARE containing proinflammatory cytokinesTNFα and GM-CSF.[122,124] This aspect of mRNA stability con-trol would therefore represent an undesirable effect of p38 inhi-bition in inflammatory contexts.

In addition to effects on TTP, there are a number of reportsdocumenting p38 MAPK-dependent responses that could be anti-inflammatory. For example, the MAPK-interacting kinases(MNKs), MNK1 and MNK2, repress translation by increasing thephosphorylation status of eukaryotic initiation factor 4E (eIF4E),a factor that is important in growth and proliferative respon-ses.[125] Likewise, hsp27 is implicated in translational repression,in this case by interacting with eIF4G to facilitate dissociation ofcap-initiation complex.[126] Similarly, the p38 downstream ki-nase (PRAK), is reported to act as a negative modulator of Rassignalling,[127] whilst activation of MKK3 and p38 MAPK po-tently repressed MEK1 and ERK1/2 activity as well as reducingmatrix metalloproteinase 1 (MMP-1) gene expression.[128] Thesenegative feedback effects were found to depend on p38-mediatedactivation of the phosphatases PP1 and PP2A. Likewise, the‘anti-inflammatory’ cytokine, IL-10, (which is induced in inflam-mation situations, for example, LPS-treated monocytes), has thecapacity to down-regulate inflammatory responses, possibly in

part by inhibition of p38 MAPK, and is itself potently down-regulated by inhibition of p38 MAPK.[129,130]

6. The p38 MAPK in Inflammation and Asthma

Evidence for a role of the p38 MAPK in inflammation is welldocumented, and, like corticosteroids, it appears that the repres-sion of cytokine and other inflammatory genes by p38 MAPKinhibitors is primarily responsible for the anti-inflammatory ef-fect of these compounds.[8,131] For example, roles for p38 MAPKhave been reported in the expression of numerous inflammatoryproteins including: TNFα, IL-1β, IL-2, IL-6, IL-8, GM-CSF andCOX-2. In addition, the involvement of p38 MAPK in the induc-tion of intercellular adhesion molecules (ICAMs), vascular celladhesion molecules (VCAMs), inducible nitric oxide synthase(iNOS), as well as playing roles in responses such as the differ-entiation, proliferation, and chemoattraction of various inflam-matory cells in response to cytokines such as IL-5, GM-CSF,granulocyte colony-stimulating factor (G-CSF), and eotaxin, allsupport a beneficial effect of p38 inhibitors in asthma (see figure4).[131]

6.1 p38 MAPK in Eosinophils

One prominent feature of asthmatic inflammation is the infil-tration of eosinophils into the lung. Eosinophils are bone marrow-derived inflammatory cells that degranulate to release cytotoxicproteins and oxygen radicals and thereby promote tissue damageand remodelling of the airways.[132] Consequently, the role ofinflammatory chemokines and cytokines in the recruitment ofeosinophils from the blood and their activation in the lung is ofmajor importance in asthmatic inflammation.[132] Cytokines suchas IL-3, IL-5 and GM-CSF promote eosinophil differentiation inthe bone marrow and lead to blood eosinophilia. These circulat-ing eosinophils are then recruited to the lung by chemoattractantchemokines such as eotaxin. Once in the lung, eosinophil activa-tion and survival is promoted by factors including GM-CSF, IL-5and eotaxin. However, eotaxin-dependent chemotaxis and releaseof eosinophil cationic protein (ECP) were markedly inhibited bythe p38 MAPK inhibitor, SB 202190,[133] whilst SB 203580 en-hanced eosinophil apoptosis and was a potent inhibitor of theLTB4-induced respiratory burst.[134,135] In an independentstudy,[136] SB 202190 inhibited eosinophil differentiation, de-granulation, as indicated by eosinophil-derived neurotoxin(EDN), and cytokine release suggesting that the p38 MAPK playsa central role in multiple eosinophil effector functions. Consistentwith these findings, a second generation p38 inhibitor, SB239063, induced eosinophil apoptosis, even in the presence ofIL-5, and markedly reduced mouse and guinea pig ovalbumin-

120 Newton & Holden


induced lung eosinophilia as well as leukotriene-induced guineapig lung eosinophilia.[22]

6.2 p38 MAPK in Airway Epithelial and Goblet Cells

The epithelial cell layer of the airways extends from the lar-ynx to the terminal bronchioles and is the primary site of contactwith airborne allergens, irritants, pathogens and other pro-inflammatory agents. This layer is biosynthetically active andacts as a source of multiple inflammatory cytokines, chemokines,prostanoids and other mediators.[137] In addition, within the epi-thelium, goblet or type II cells are responsible for elevated mucussecretion in asthma and may be meta- or hyperplasic . Analysisof the effect of SB 203580 in human bronchial epithelial cell linesrevealed a marked inhibition of IL-1β-, TNFα- and platelet acti-vating factor (PAF)-induced production of IL-8, RANTES (reg-

ulated on activation normal T cell expressed and secreted) andGM-CSF.[138,139] Likewise, prostaglandin E2 (PGE2) release fromthe pulmonary epithelial cell line A549 was inhibited by SB203580.[64] Similarly, other studies have indicated roles for thep38 MAPK in the IL-17-induced release of neutrophil-mobilisingcytokines,[140] whilst preliminary findings indicate that p38 in-hibitors may be effective in preventing MUC5AC (mucin) geneexpression.[141]

6.3 p38 MAPK in Airway Smooth Muscle

As well as being a source of proinflammatory cytokines andmediators, including RANTES and eotaxin,[142] there is ampleevidence supporting a positive correlation between increasedsmooth muscle mass and the pathogenesis of asthma.[143] Suchremodelling events are in part due to hyperplasia and hypertrophy

Activation of p38

Eosinophils Epithelialcells

Smoothmuscle

Endothelialcells

Mastcells

Lymphocytes Macrophage/monocytes

Neutrophils

Upregulationof degranulationand chemotaxis

Increased productionof RANTES, GM-CSFmucins and IL-17

Increased cellmotility andproinflammatorycytokine production

Increasedproductionof IL-8

Increased cellmigration andproduction of IL-8

Upregulation ofIL-5 and IL-13

Promotes monocytedifferentiation, monocytechemotaxis and productionof MIP-1α, MIP-2, TNFα,COX-2 and GM-CSF

Promotes IL-8 stimulateddegranulation, shape changeand production of secondary,tertiary and secretory granules.Inhibits CD11b/CD18 function

Increase in severity of asthma

Decrease in inflammatorymRNAs stability viaproduction of TTP

Inhibition of translationthrough interaction withthe initiation factorseIF4G and eIF4E

Repression of the Ras,MEK1 and ERK1/2signalling pathways

Decrease in cell profilerationthrough inhibition of cyclinD1 in smooth muscle cells

Reduction of MMPgene expressionin fibroblasts

Production of IL-10 andIL-12 in macrophagesand monocytes

Activation of p38

Decrease in severity of asthma

a

b

Fig. 4. Effects of p38 activation in asthma. The scheme shows various p38 mitogen-activated protein kinase (MAPK)-dependent events that may be expected to impact oninflammation and asthma severity. In panel a, the inflammatory effects of p38 MAPK on cells relevant to airway inflammation and asthma are shown. Panel b depicts a number ofevents when the actions of p38 MAPK may be expected to be anti-inflammatory or helpful to the resolution of asthma. Overall this scheme demonstrates a bias towards p38-dependentevents that increase asthma severity and would be reduced by inhibitors of the p38 MAPK. COX-2 = cyclo-oxygenase 2; ERK = extracellular regulated kinase; GM-CSF =granulocyte-macrophage colony-stimulating factor; eIF4G = eukaryotic initiation factor 4G; eIF4E = eukaryotic initiation factor 4E; IL = interleukin; MEK1 = MAPK/ERK kinase 1;MIP-1α = macrophage inflammatory protein-1α; MIP-2 = macrophage inflammatory protein-2; MMP = matrix metalloproteinase; mRNA = messenger RNA; RANTES = regulatedon activation normal T cell expressed and secreted; TNFα = tumour necrosis factor-α; TTP = tristetraproline.



of the airway smooth muscle, and cytokine or growth factors suchas TGFβ and platelet-derived growth factor (PDGF) are stronglyimplicated. In this context, the inhibition of p38 MAPK mayreduce cell migration in response to PDGF, IL-1β and TGFβ.[144]

However, in primary airway smooth muscle cells the expressionof cyclin D1, which is required for cell cycle progression, is ac-tually increased by inhibition of p38 MAPK.[145] Furthermore,this conflicting observation is supported by the recent finding thatlung myofibroblast proliferation is also increased by p38 MAPKinhibitors.[146] Conversely, p38 inhibitors have the ability to re-press biosynthetic functions of airway smooth muscle as indi-cated by the inhibition of IL-4- and IL-13-induced eotaxin re-lease.[147]

6.4 p38 MAPK in Endothelial Cells

The influx of inflammatory cells to the lung is regulated toa large extent by vascular endothelial cells. These cells expressvarious surface adhesion molecules, including ICAM-1, that arenecessary for inflammatory cell attachment and then diapedesisfrom the blood to the site of inflammation.[148] In addition, theendothelium is important in inflammatory cell recruitment via theproduction of cytokines, chemokines and other mediators andmany of these responses are repressed by inhibitors of the p38MAPK. For example, the neutrophil and monocyte chemo-attractants, IL-8 and monocyte chemotactic protein-1 (MCP-1),respectively, are secreted by human endothelial cells followinginflammatory stimulus and these responses are attenuated by in-hibitors of p38 MAPK.[149-151] In addition, p38 MAPK is impli-cated in both the expression of ICAM-1 on endothelial cells andin the ICAM-1-dependent cytoskeletal arrangement that occursfollowing ICAM-1 ligation.[152,153] Likewise, IL-6 production,prostaglandin production and MMP expression are also repressedby p38 inhibition, suggesting that not only is the recruitment ofinflammatory cells to the endothelial layer prevented by p38 in-hibition, but also factors that are involved in cell efflux and oe-dema formation are also reduced.[154]

6.5 p38 MAPK in Mast Cells

Mast cells accumulate in the mucosal epithelial layer of thelung and are central to allergic reactions. When activated, forexample by FCεRI aggregation following binding of multivalentantigen to IgE present on the cell surface, mast cells release var-ious mediators including the proinflammatory cytokines, IL-1β,TNFα and IL-6 as well as the pro-asthma cytokines, IL-5, IL-4and IL-13.[155] In addition, activation and degranulation of mastcells releases potent bronchoconstrictors including histamine andleukotrienes.[155] Although mast cells migrate in response to chemo-

kines, recruitment to the inflamed areas is also partly antigen/FCεRI-dependent and may be susceptible to p38 MAPK inhibi-tion.[156] In addition, the p38 MAPK is implicated in the adeno-sine A2B receptor-mediated release of IL-8 by mast cells.[157]

Thus, these data are indicative of a broad positive effect of p38inhibition on various pro-asthma effects of mast cells.

6.6 p38 MAPK in Lymphocytes

Activation of T-lymphocytes of the Th2 subtype and the sub-sequent release of a Th2 cytokine profile is crucial in the devel-opment of eosinophil responses due to the potent eosinophilgrowth, differentiation, activation and chemotactic properties ofthe Th2 cytokine, IL-5.[132] In cell based studies, the release ofIL-5, but not IL-2, IL-4 or IFNγ, from human T cells stimulatedwith a variety of stimuli was strongly inhibited by SB 203580 insome studies,[158] but not in others.[159] Another pro-asthmacytokine that is released by T cells and is inhibited by SB 203580is IL-13.[160] As well as being a classical Th2 type cytokine, IL-13is essential in signalling to B-lymphocytes to induce immuno-globulin gene rearrangement and production of IgE.[161] In addi-tion to indirect effects occurring via IL-13, p38 is also implicatedin CD40-dependent-IgE isotype switching in B lymphocytes.[162]

Taken together these data suggest an important role for the p38MAPK in various T and B cell effector responses that are impor-tant in asthma and allergy.

6.7 p38 MAPK in Monocyte/Macrophage Cells

Whilst the role of macrophage cells is primarily in host de-fence and innate immunity, the immunomodulatory properties ofthese cells also makes them important in inflammatory diseasessuch as asthmatic inflammation and COPD.[163] Thus the recruit-ment of monocytes from the blood, activation, and subsequentbiosynthetic ability may be significant in diseases such as asthmaand COPD.

In addition to indirect effects on monocyte recruitment, forexample, via effects on adhesion molecule or chemokine expres-sion from structural cells, the p38 MAPK pathway also impactson various monocyte effector functions. Thus, L-790070, a novelp38 MAPK inhibitor (table I), prevented serum-induced mono-cyte differentiation and chemotaxis.[164] In addition chemotacticresponses to macrophage inflammatory protein-1α (MIP-1α), MCP-1 and N-formyl-methionyl-leucylphenylalanine (fMLP) were alsosensitive to inhibition by L-790070, whereas macrophage phago-cytosis remained unaffected.[164] Similarly, the novel p38 MAPKinhibitor, M39, prevented release of TNFα and macrophage in-flammatory protein-2 (MIP-2) from isolated human macrophagecells.[165] Again expression of COX-2 and GM-CSF from mono-

122 Newton & Holden


cytes has been shown to be at least partly p38-dependent indicat-ing the wide role of this pathway in this cell type.[129,166] Con-versely, p38 MAPK may also play a role in anti-inflammatoryeffects of macrophages. Thus, expression of the anti-inflammatorycytokine, IL-10, was shown to be up-regulated by p38 in murinemacrophages.[167] Furthermore, p38 MAPK is thought to be in-volved in the induction of IL-12 production by macrophages, andsince IL-12 is essential for the development of the Th1 immuneresponse, inhibition of p38 in the macrophage may cause an in-crease in the Th2 immune response therefore exacerbating airwayinflammation.[168]

6.8 p38 MAPK in Neutrophils

In mild to moderate asthma, it is eosinophils, mast cells andT lymphocytes that are primarily believed to contribute to airwayinflammation, whereas diseases such as COPD are generally as-sociated with neutrophilic inflammation.[72] However, until a re-cent study by Jatakanon 1999[169] there was little data regardinginflammatory cell influx in patients with severe asthma. Thisstudy provides evidence that, in addition to eosinophilic infiltra-tion, patients with severe asthma may also show a substantialinflux of neutrophils to the lung.[169] Thus, the effects of p38MAPK inhibitors on neutrophil functions should be consideredin the context of asthma as well as in COPD. One indirect effectof p38 inhibitors on neutrophil numbers is likely to occur via theinhibition of the release of the chemotactic factor IL-8 from struc-tural cells such as airway epithelial cells,[149,150] for example,induced by the proneutrophilic cytokine IL-17,[140] endothelialcells,[170] smooth muscle or other structural cells, as well as fromvarious inflammatory cells.[171,172] Moreover, neutrophil chemo-taxis may also be reduced by direct effects of p38 inhibitors onneutrophils.[173] Using a novel in vivo chemotaxis assay, the abil-ity of murine leucocytes (neutrophils) to chemotax to and emi-grate from, but not roll or stick to, vessels in response tokeratinocyte-derived cytokine (KC) [the mouse homolog ofgrowth regulated gene (GROα) that acts via CXCR2, the mousehomolog of IL-8RB], was impaired by p38 MAPK inhibitors.[174]

In addition, the actual ligation and cross linking of L-selectins onthe neutrophil activates p38 MAPK making the neutrophil moresusceptible to IL-8-stimulated degranulation.[175] This responsewas blocked by the potent p38 MAPK inhibitor L-790070.[23]

Taken together these findings are consistent with the reductionsin lung neutrophilia, cytokine output and MMP-9 expression fol-lowing administration of SB 239063 in a rat model of lung in-jury.[172]

6.9 p38 MAPK in Neurogenic Inflammation

In addition to the classical inflammatory process, it is nowreasonably well established that the nervous control of the air-ways and the production of neuropeptides also plays a significantpart in the pathophysiological processes of airway diseases.[176]

Some of the most potent neuroimmunomodulatory mediators aremembers of the tachykinin family. Two members of this family,substance P (SP) and neurokinin A (NKA), have been localisedto sensory nerves within the airway and elicit downstream effectsvia neurokinin 1 (NK1) and NK2 receptors, respectively.[176]

These responses include airway smooth muscle constriction, va-sodilation, plasma exudation from bronchial vessels, increasedsubmucosal gland secretion, increased ion transport, ciliary beat-ing and release of prostanoids in the airway epithelium. In addi-tion, increased acetylcholine release and ganglionic transmissionin nerves, increased mediator release from macrophages and in-creased collagen secretion from fibroblasts may also be ob-served.[177] Whilst the actual role of p38 MAPK in these pro-cesses is poorly understood, studies in an astrocytoma cell linehave shown that SP activates the p38 MAPK and IL-6 expressionin a manner that was prevented by the p38 inhibitor, SB202190.[178] Since SP can also induce IL-1β, TNFα and IL-6synthesis, there is a real possibility that p38 MAPK inhibitorsmay prove effective against neurogenic inflammation.[179]

6.10 p38 MAPK in Vivo

During their initial characterisation, CSAIDs were shown tohave anti-inflammatory potential in a variety of in vivo models.[131]

Whilst these studies demonstrate that p38 inhibitors are well tol-erated by animals, the models used were mostly for rheumatoidarthritis. More recently, RWJ 67657 (table I) was found to inhibitTNFα production from LPS-treated human peripheral bloodmononuclear cells and both LPS-injected rats and mice.[180]

These findings are consistent with the previous finding that thedownstream kinase, MAPKAP-K2 is essential for the inductionof TNFα synthesis, and knock-out mice that do not have a func-tional copy of this gene showed markedly enhanced survival inresponse to LPS-induced toxic shock.[119] Similarly, and of morerelevance to asthma, SB 203580 was shown to inhibit by up to95% the production of LPS- and ovalbumin-induced TNFα andIL-1β in bronchoalveolar lavage (BAL) fluid from brown Nor-way rats.[181,182] However, the induction of both eosinophilia andneutrophilia by ovalbumin was not apparently affected by SB203580 and therefore the role of p38 MAPK in these events maybe open to question.[182]

Further studies in brown Norway rats using the more potentand selective p38 inhibitor, SB 239063, revealed a marked re-



pression of ovalbumin-induced eosinophil and lymphocyte re-cruitment to the lung.[183] By contrast, using Balb/c mice in anovalbumin sensitisation and rechallenge model revealed no effectof L-790070 on eosinophil numbers or cytokine expression inBAL fluid or on goblet cells.[184] However, in this study a markedreduction in neutrophil numbers and a lowering of methacholinesensitivity was observed. In addition, inhibition of p38 MAPKwas found to reverse the ovalbumin-induced increase in mucussecreting cell numbers. Similarly, atmospheric pollutants, irri-tants and oxidative stresses, such as ozone, are known to exacer-bate various lung diseases including asthma and COPD. To ex-amine these effects an acute model using ozone inhalation byBalb/c mice to increase the expression of the neutrophilic cyto-kine, KC, and IL-6 and elevate BAL neutrophils was estab-lished.[185] In this model the increase in BAL neutrophils wassignificantly attenuated by prior administration of SB 239063suggesting that such compounds may be effective in both chronicand acute disease.

In rats, the pharmacokinetic properties of SB 203580 reveala low and variable oral bioavailability, evidence of non-linearelimination and moderate to high amounts of drug bound toplasma proteins.[186] This raises the possibility that in vivo phar-macology studies employing SB 203580 could be flawed. Morerecently, second generation compounds, such as SB 242235,showed improved preclinical pharmacokinetic properties includ-ing a high bioavailability (50–100%) and low to nonexistent sec-ondary metabolism suggesting that these compounds may bemore effective in clinical studies.[187,188] At this point, a note ofcaution is appropriate since targeted disruption of the p38 genein mice results in embryonic lethality due to placental de-fects.[189,190] In addition, mice that develop for longer show se-vere anaemia due to deficient erythropoietin expression.[189] Fur-thermore, the wide variety of tissues that express p38 isoformsraises the possibility that these kinases may play a number of asyet unsuspected roles. In this respect, strong p38α MAPK immu-noreactivity was found in numerous structures within the brainraising the possibility that p38 MAPK may be important in brainfunction.[191] Finally, as a salient reminder as to the possible ad-verse consequences of drugs that are potentially immunosuppres-sive, it has been noted that despite displaying an enhanced resis-tance to endotoxic shock, MAPKAP-K2 deficient mice show anincreased susceptibility to infection by Listeria monocyto-genes.[192]

7. Conclusion

In the context of inflammatory pulmonary diseases such asasthma and COPD there are a number of different targets for p38

within the airways and lungs (see figure 4). The involvement ofp38 MAPK in many of these processes has been suggested byvarious studies looking at cell types relevant to airway disease.In addition, p38 MAPKs have been shown to be involved in theregulation of inflammatory gene expression at various levels in-cluding transcriptional control, particularly of NF-κB and AP-1,as well as posttranscriptional and translational control. These di-verse roles for p38 MAPK are reminiscent of corticosteroids,which also show inhibition of both AP-1 and NF-κB-dependenttranscription as a means of anti-inflammatory action, andstrongly suggest that the inhibition of p38 MAPK may also pro-vide anti-inflammatory benefits.[8,66] Whilst the overall picturesuggests that inhibition of p38 MAPK may be broadly beneficialin inflammatory diseases such as asthma, there are a number offunctions of p38, which if inhibited, could be unhelpful to diseaseresolution and could lead to a complex phenotype in response tothe therapeutic use of p38 MAPK inhibitors. In addition, the find-ings that p38α knock-out mice show a lethal phenotype and thefact that various structures within the brain show high levels ofp38α expression indicates the need for extreme caution.

A number of compounds are currently undergoing or aboutto undergo phase I (SB 281832) and phase II (SCIO-469) clinicaltrials. The Vertex Pharmaceuticals compound, VX-745, has ac-tually completed both phase I and phase II clinical trials for rheu-matoid arthritis and is now being discontinued in favour of thenewer compounds, VX-702 and VX-850, which have now com-menced phase I and preclinical trials. In terms of treatments forasthma, or other airway diseases the development of formulationssuitable for inhalation is currently underway. Overall, we believethat inhibitors of p38 MAPK show some promise in the treatmentof inflammatory diseases such as asthma.

Acknowledgements

Neil Holden is funded by the MRC and Novartis Pharmaceuticals UK Ltd.

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Correspondence and offprints: Dr Robert Newton, Department of BiologicalSciences, University of Warwick, Coventry, CV4 7AL, UK.E-mail: [email protected]



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Inhibitors of p38 Mitogen-Activated Protein Kinase