key: cord-0852182-fquz3scq
authors: Newton, Robert; Holden, Neil S.
title: New aspects of p38 mitogen activated protein kinase (MAPK) biology in lung inflammation
date: 2006-05-31
journal: Drug Discovery Today: Disease Mechanisms
DOI: 10.1016/j.ddmec.2006.02.007
sha: 1a6d6b882b0faaa0e6fd78c89e5bd4f73bacc64a
doc_id: 852182
cord_uid: fquz3scq

Lung inflammation features in asthma, chronic obstructive airways disease (COPD), acute respiratory distress syndrome (ARDS), cystic fibrosis (CF) and others. Whilst in asthma anti-inflammatory glucocorticosteroids are generally effective, certain individuals are steroid resistant and in COPD, ARDS and CF, as well as disease exacerbations caused by infection, there seems little benefit. We summarise recent advances in p38 mitogen activated protein kinase (MAPK) biology and document beneficial and possibly detrimental effects in respect of lung inflammation. Section editors: Maria Belvisi – Imperial College School of Medicine, London, UK Stuart Farrow – GlaxoSmithKline, Stevenage, UK

In asthma, COPD, ARDS and other inflammatory diseases, the upregulation of cytokines, chemokines and other proteins leads to the recruitment and influx of inflammatory cells. The p38 mitogen activated protein kinase (MAPK) plays a key role in these processes via ligands binding to receptors or cellular stresses (Box 1). Small G-proteins then activate MAPK kinase kinases (MAP3K), which phosphorylate, and activate, the MAPK kinases (MKKs) (Fig. 1) . MKK6 activates all four (a, b, g, d), whereas MKK3 activates the a and b p38 MAPK isoforms. Downstream targets are numerous and play roles in the regulation of inflammation via transcriptional, post-transcriptional, translational and other targets (Fig. 1) .

Finally, in understanding p38 MAPK biology, it is important to appreciate that the commonly available inhibitors are selective for p38a and p38b, not p38g and p38d, and our current knowledge of targets ( Fig. 1 ) and responses (Tables 1 and 2) reflects this.

Activation of p38 MAP kinases by cellular stresses and inflammatory cytokines, such as TNFa and IL-1b, is well established (Fig. 1) [1, 2] . In addition, newer stimuli, for example, the proinflammatory cytokine, IL-17, which induces IL-8 synthesis (see [2] ), IL-18, which primes neutrophil functions [3] , or IL-25, a novel Th2 cytokine that upregulates cytokine and chemokine expression from eosinophils [4] , are continually being described (Box 1). However viruses and bacteria, as the causative agents of pulmonary diseases, along with their products, also activate the p38 MAPK and are increasingly coming to light as principal causes of exacerbations in asthma and COPD [2, 5] .

Thus respiratory viral infections, for example, human rhinovirus (HRV) and respiratory syncytial virus (RSV), upregulate the expression of multiple cytokines (IL-1, TNFa, IL-6, G-CSF, GM-CSF) and chemokines (IL-8, ENA-78, GROa, RANTES) in epithelial cells, as well as ICAM-1 in endothelial cells, via the p38 MAPK pathway [5] [6] [7] [8] . Activation of p38 MAPK by influenza virus (IV), including the H5N1 'bird flu' strain, suggests a similar induction of cytokines and chemokines [9] . These effects are likely to be mediated via the MAP3K, apoptosis signal-regulating kinase (ASK) 1, which is also responsible for IV-induced apoptotic cell death [10] . Furthermore, the finding that double stranded RNA (dsRNA), which acts via the dsRNA-dependent protein kinase (PKR) and mimics responses to many RNA viruses including RSV, HRV and IV, suggests that these are general responses to viral infection [5] . In addition, HRV infection of alveolar macrophage and monocytic cells leads to p38 and activating transcription factor (ATF)-2 phosphoryation as well as the induction of MCP-1 expression [11] . Therefore, despite not affecting HRV replication, IV infection, or IV and severe acute respiratory syndrome (SARS) viral protein synthesis, these data indicate the therapeutic potential of targeting p38 MAPK in epithelial and macrophage cells in the context of virus-induced inflammation and apoptosis [6, 9, 10, 12] .

Similarly, the bacterial product, lipopolysaccharide (LPS), is a potent inducer of p38 MAPK and in LPS-induced ARDS, this pathway plays a role in both bronchoconstriction and neutrophil recruitment [2, 13] . Numerous other bacterial pathogens also induce pulmonary inflammatory responses via p38 MAPK. For example, Burkholderia pseudomallei, the causative agent of melioidosis, activates p38 MAPK and this appears necessary for epithelial cell invasion [14] . Burkholderia cenocepacia, a pathogen that causes fatal pulmonary disease in the immunocompromised and CF sufferers, induces expression of the bradykinin B1 kinnin receptor, and presumably other inflammatory mediators, via a p38-dependent mechanism in fibroblasts [15] . Likewise, Streptococcus pneumoniae, the predominant cause of community-acquired pneumonia, and a major cause of death by infectious disease in industrialised countries, induces IL-8 expression via the p38 pathway [16] . This is also true for cell fractions and lipopeptide, from nontypeable Haemophilus influenzae, which is a major cause of COPD exacerbation [17] .

In addition, several novel inflammatory compounds, including pollutants, such as ultrafine carbon particals and cigarette smoke [18, 19] , lipid mediators, such as leukotriene C 4 (LTC 4 ) and LTD 4 [20, 21] , as well as mechanical stretch or cholinergic stimulation [22] , all activate the p38 MAPK and might therefore contribute to inflammatory processes. Interestingly, the response to cigarette smoke was synergistically increased by heat-inactivated bacteria suggesting the possibility of combinatorial effects in diseases such as COPD [19] .

The p38 MAPK regulates gene transcription via phosphorylation of numerous transcription factors (Fig. 1 ). In addition, the activity of AP-1, a major positive regulator of inflammatory genes, is also enhanced by increasing the expression of the constituent proteins, c-Jun and c-Fos. In this case, p38dependent phosphorylation of tenary complex factors (TCFs) promotes interaction with serum response factor (SRF) to drive transcription from serum responses elements (SREs), such as are found in the c-fos promoter [1, 2] . Alternatively, effects mediated via adenosine-and uridine-(AU)-rich elements (ARE) in the 3 0 untranslated region (UTR) of c-fos and cjun can also enhance expression (see below).

p38 MAPK can also act downstream of transcription factor DNA binding [2] . Thus, p38 MAPK potentiates the transcriptional competency, not DNA binding, of the inflammatory transcription factor, NF-kB, via processes that may also determine differential responsiveness of NF-kB-dependent genes [2] . Similarly, p38 inhibitors prevented the S. pneumoniaedependent induction of IL-8 and GM-CSF from bronchial epithelial cells by blocking NF-kB-dependent transcription and phosphorylation, but not nuclear transclocation or recruitment to the promoter [16] .

Glucocorticosteroid control of asthma occurs via the glucocorticoid receptor (GR). However, in some patients clinical utility is limited by steroid-insensitivity, a phenomenon that may involve phosphorylation of GR by MAPKs and reduced anti-inflammatory ability (see [2] ). Certainly, p38-dependent phosphorylation of GR diminishes GR-dependent transcriptional responses [23] , which given a role for steroid-inducible Growth factors EGF, PGDF, TGFb

Neurotransmitters, kinins and others Substance P, bradykinin, acetylcholine (methacholine) adenosine, fMLP

Platelet activating factor (PAF), leukotrienes (LTC 4 , LTD 4 )

Oxidative stress (ROS), stretch, hyperosmolarity, hypoxia

Bacterial products (LPS, peptidoglycans), Burkholderia pseudomallei, Burkholderia cenocepacia, Haemophilus influenzae, Pseudomonas aeruginosa, Streptococcus pneumoniae, Influenza virus (IV), respiratroy synccytial virus (RSV), adenovirus, human rhinovirus (HRV), SARS virus.

Diesel exhaust/carbon particulates, cigarette smoke, CO (carbonmonoxide), thrombin, ECM components, ICAM-1 ligation, IgE/FCeRI ligation, CD40 ligation, serum, phorbol esters, dsRNA p38 MAPK activating stimuli listed are taken from the current review and references [1, 2] . The present list is therefore not exhaustive. genes in the anti-inflammatory actions of glucocorticosteroids [24] , points to an involvement in steroid resistance.

The characterisation of cytokine-suppressive anti-inflammatory drugs (CSAIDs) (p38 inhibitors) revealed inhibition of cytokine biosynthesis via post-transcriptional and translational mechanisms [1, 2] . This involved the downstream kinase, MAPK activated protein kinase 2 (MAPKAP-K2) and is particularly relevant for genes, such as TNFa, that contain AREs in their 3 0 UTRs (Table 3 ) [24] . Indeed, many inflammatory genes contain one or more ARE (Table 3) , and p38dependent mRNA stabilisation therefore regulates inflammatory and virally induced gene expression [7, 24] . Despite the identification of numerous ARE-binding proteins (ARE-BPs) [25] (Fig. 2) , there is considerable uncertainty as to targets of the p38 pathway [26] . This is being said, that binding of heterogenous nuclear ribonuclear protein (hnRNP) A0 to the TNFa, COX-2 and MIP-2 3 0 UTRs is MAPKAP-K2-dependent, blocked by the p38 inhibitor, SB203580, and correlates with mRNA stability [25] . Similarly, binding of hnRNP A1 to the TNFa ARE increases following phosphorylation by MNK [27] , kinases which are also implicated in translation via phosphorylation of the eukaryotic initiation factor 4E (eIF4E) (see [2, 24] ) (Fig. 2) . Furthermore, Mnk knock-down, or pharmacological inhibition, reduces TNFa expression and supports a role for this pathway [27] . Further complexity is introduced as p38-dependent stabilisation of certain AREcontaining mRNAs (e.g. COX-2, TNFa) occurs via blocking deadenylation [28] to promote both mRNA stability and translation which are themselves coupled to poly-A tail length (Fig. 2) . Thus poly-A tail shortening reduces translation efficiency, precedes mRNA degradation and can be targeted by the p38 MAPK to regulate inflammatory gene expression [24, 28] (Fig. 2) .

The p38 MAPK is subject to inhibition by three main classes of phosphatase [24] . In this context, the dual-specificity phosphatase, MAPK phosphatase 1 (MKP-1), is rapidly induced by pro-inflammatory stimuli, for example LPS on alveolar macrophage [29] ; to terminate p38 signalling [24] . MKP-1 is also induced by glucocorticosteroids and this illustrates an anti-inflammatory mechanism by which steroid-dependent transcription prevents biosynthesis (transcriptional, posttranscriptional and translational in Fig. 1 ) of inflammatory mediators [24, 29] . Furthermore, cAMP-elevating agents also induce MKP-1 raising the possibility of a role in the antiinflammatory effects of long acting b 2 -agonists and other cAMP-elevating drugs [24] . One surprising consequence of reduced p38 activity, is that the induction of Toll-like receptor-2 (TLR2) by nontypeably H. influenzae (NTHi) is subject to p38-dependent feedback inhibition such that NTHi infection in the presence of glucocorticosteroids enhances TLR2 expression and signalling to increase the release of cytokines and chemokines [30] . Thus by inducing MKP-1 and inhibiting p38, glucocorticoids can enhance inflammatory responses to certain infections and this could also occur in the context of p38 inhibitors.

As noted above, binding of ARE-BPs to 3 0 UTRs imparts considerable regulatory control. Tristetraprolin (TTP) is one such protein, which is responsible for mRNA destabilisation and who's deletion elevates TNFa expression and leads to various inflammatory disorders [24] . This acute phase gene is induced by pro-inflammatory stimuli and provides negativefeedback control [24] . Therefore, the inhibition of proinflammatory pathways could, by preventing TTP expression, stabilise and enhance the expression of ARE containing genes. In this context, the p38 pathway, acting via MAP-KAP-K2 and the TTP ARE, stabilises TTP mRNA and p38 inhibition profoundly reduces TTP expression [31] . Furthermore, TTP destabilising activity might require p38-dependent phosphorylation and provides additional evidence that effects of the p38 MAPK on TTP might be desirable in inflammation [24] .

Further key modulatory roles include the p38-dependent expression of the anti-inflammatory cytokine, IL-10, and the p38-dependent induction of suppressor of cytokine signal- . p38 MAPK targets the ARE-BPs, hnRNP A0 and A1, via MK2 and MNK, respectively, and this may play a role in mRNA stabilisation. Other proteins that may be targeted by the p38 MAPK include PABP and other ARE-BPs. In addition, p38 MAPK may also target a deadenylase to prevent loss of the poly-A tail and promote mRNA stability and translation. This along with phosphorylation of eIF4E may facilitate association between the poly-A and the cap structure, an event that could be promoted by ARE-BPs, and lead to enhanced efficiency of translation. Finally, the p38 MAPK, acting via MK2, promotes TTP expression and also activates TTP by phosphorylation to exert negative feedback control and destabilisation of ARE containing mRNAs. Other abbreviations: AUF, AU-binding factor; BRF-1, butyrate-response factor 1; hn RNP, heterogenous nuclear ribonuclear protein; TIA-1, T cell-restricted intracellular antigen-1 (TIA1-); TIAR, TIA-related protein.

ling 3 (SOCS3) by IL-4, which might be important in Th2dependent diseases such as asthma . As SOCS3 limits signalling via gp130 cytokine receptors, the inhibition of p38 MAPK could lead to exaggerated responses and amplification of Th2-dependent disease.

Many of the responses above, for example, release of and responses to IL-8 [2, 3, 16, 17] , suggest a major effect on neutrophillic disease. Neutrophil influx is usually a hallmark of obliterative bronchiolitis following lung transplantation and using an in vivo rat model, a p38 inhibitor was shown to dramatically reduce inflammatory cytokines, tracheal occlusion and organ rejection [33] . This is consistent with data from LPS-challenged mice in which TNFa production was reduced by SB239063, a potent second-generation p38 inhibitor ( [2] and see refs therein). Likewise in an ovalbumin (OVA) sensitisation and challenge mouse model, a p38 inhibitor again prevented neutrophil increases in the bronchoalveolar lavage (BAL) fluid [34] . Interestingly, whilst increased cytokine levels (for IL-4, -5, -12, -13 or interferon-g) and goblet cell hyperplasia were unaffected, mice treated with p38 inhibitor revealed significantly decreased airways hyperreactivity (AHR) [34] . Similarly, OVA-induced eosinophilic inflammation in both mice and guinea pigs was also prevented by SB239063 (see [2] ). This compound reduced LTD 4induced eosinophilia and promoted eosinophil apoptosis suggesting a beneficial effect in eosinophilic diseases, such as asthma. This conclusion also receives support from an OVA-induced mouse asthma model in which antisense oligonucleotides to p38 MAPK prevented pulmonary eosinophilia, AHR and mucus hypersecretion [35] . In addition, lung oedema is frequently associated with disease. This is promoted by the down-regulation of the epithelial sodium channel, ENaCa, to reduce water and ion transport into the tissues. As this process requires p38 MAPK, p38 inhibitors could aid the control of lung oedema [36] .

In terms of infection and disease exacerbation, epithelial cells from COPD patients show enhanced, as well as additional (versus non-COPD patient), p38 MAPK-dependent inflammatory responses to H. influenzae [37] . Likewise CF tissues showed enhanced responses to Pseudomonas aeruginosa LPS via increased IL-8 release and neutrophil migration and therefore suggest a benefit from p38 inhibition in CF inflammation [38] . One possible worry in respect of p38 inhibition in infection relates to the ability of DNA containing unmethylated CpG, as occurs in the context of bacterial infection, to induce strong Th1-type immune responses and reduce the development of Th2 allergic asthma in a mouse model [39] . This effect, which might be a part of immunological education, requires the p38-dependent release of the pro-Th1 cytokine, IL-12, from alveolar macrophage. Thus the use of p38 inhibitors to combat inflammation in infection could subsequently enhance the development of allergic disease. Another, frequently fatal disease with relatively rapid onset is idiopathic pulmonary fibrosis (IPF). This is poorly responsive to current treatments and is characterised by irreversible lung fibrosis, which is now reported to involve the p38 MAPK [40] . Given a role of p38 MAPK in the expression of growth factors and fibroblast functions [2] , it is possible that p38 inhibition may prove to be of therapeutic benefit.

The above examples reveal a critical role of the p38 MAPK in regulating inflammatory gene expression and suggest a key role in pulmonary disease. These effects occur by various transcriptional, post-transcriptional and translational mechanisms and the data presented supports the potential use of p38 inhibitors in controlling inflammatory responses. The fact that anti-inflammatory glucocorticoids target the p38 pathway, via the induction of MKP-1, supports the pharmacological rationale for targeting p38 MAPK in inflammation. Furthermore, the finding that the p38 MAPK targets GR to reduce responsiveness raises the possibility that p38 inhibitors could be used, not only in their own right as antiinflammatory agents, but also in conjunction with glucocorticosteroids to improve patient sensitivity to these compounds. This effect could be particularly valuable in the context of steroid resistant or insensitive patients who often remain poorly controlled, if at all, and require high dose oral corticosteroids. In this context, disease progression in COPD is poorly responsive to glucoccorticosteroids and pathogenesis involves the neutrophil. As p38 inhibitors appear effective at targeting neutrophil functions and many COPD triggers, for example cigarette smoke or particulates, promote neutrophil recruitment via the p38 pathway, inhibitors of the p38 pathway may show benefit in this disease. In addition, both viral and bacterial inflections are potent inducers of the p38 MAPK and lead to significant health issues both arising directly from infectious disease as well as from the exacerbation of conditions such as asthma or COPD. Thus in situations where the underlying infection is controllable by other means, such as antibiotics or anti-virals, it is possible that p38 inhibition may be appropriate to deal solely with the resultant inflammatory response. Notwithstanding this positive outlook, it is to be noted that p38-dependent processes also take a significant role in feedback inhibition of inflammatory genes via the expression and activation of TTP. Removal of these control processes could tend to increase inflammatory gene expression. Thus the balance between these positive and negative effects can be stimulus-and gene-specific and will only become apparent following further studies. In addition, whilst there is some evidence that infection, for example of B. pseudomallei, might require p38 MAPK, this is less certain in respect of other infectious agents and this issue still requires specific testing. Furthermore, the finding that TLR2 expression and signalling is enhanced by p38 inhibition, as a result of loss of feedback control, raises the possibility that p38 inhibitors can only be effective in the context of certain (non-TLR2-dependent) infections. Finally, loss of SOCS3 and IL-12 expression could both lead to enhanced Th2-dependent inflammatory responses and can, in time, lead to the development of elevated allergic reactions. In conclusion, these data, indicate a clear ability of p38 inhibitors to target inflammation, but certain questions remain as to potentially undesirable events that could limit the clinical utility of such compounds.

Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation

Inhibitors of p38 mitogen-activated protein kinase: potential as anti-inflammatory agents in asthma?

Physiological levels of interleukin-18 stimulate multiple neutrophil functions through p38 MAP kinase activation

Interleukin-25-induced chemokines and interleukin-6 release from eosinophils is mediated by p38 mitogenactivated protein kinase, c-Jun N-terminal kinase, and nuclear factor-kappaB

Host defense function of the airway epithelium in health and disease: clinical background

Role of p38 mitogen-activated protein kinase in rhinovirus-induced cytokine production by bronchial epithelial cells

MAPK activation is involved in posttranscriptional regulation of RSV-induced RANTES gene expression

Respiratory syncytial virus infection of human lung endothelial cells enhances selectively intercellular adhesion molecule-1 expression

mitogen-activated protein kinase-dependent hyperinduction of tumor necrosis factor alpha expression in response to avian influenza virus H5N1

ASK1 regulates influenza virus infection-induced apoptotic cell death

The role of p38 MAPK in rhinovirus-induced monocyte chemoattractant protein-1 production by monocytic-lineage cells

Phosphorylation of p38 MAPK and its downstream targets in SARS coronavirus-infected cells

Dual effects of p38 MAPK on TNFdependent bronchoconstriction and TNF-independent neutrophil recruitment in lipopolysaccharide-induced acute respiratory distress syndrome

Burkholderia pseudomallei invasion and activation of epithelial cells requires activation of p38 mitogen-activated protein kinase

Infection-induced kinin B1 receptors in human pulmonary fibroblasts: role of intact pathogens and p38 mitogenactivated protein kinase-dependent signaling

Streptococcus pneumoniae-induced p38 MAPKdependent phosphorylation of RelA at the interleukin-8 promotor

Up-regulation of interleukin-8 by novel small cytoplasmic molecules of nontypeable Haemophilus influenzae via p38 and extracellular signal-regulated kinase pathways

Ultrafine carbon particles induce interleukin-8 gene transcription and p38 MAPK activation in normal human bronchial epithelial cells

Exposure of differentiated airway epithelial cells to volatile smoke in vitro

Leukotriene C4 induces TGF-{beta}1 production in airway epithelium via p38 kinase pathway

Apoptosis signal-regulating kinase 1 in leukotriene D(4)-induced activator protein-1 activation in airway smooth muscle cells

Mechanical stretch activates nuclear factor-kappaB, activator protein-1, and mitogen-activated protein kinases in lung parenchyma: implications in asthma

Inhibition of glucocorticoid receptor-mediated transcriptional activation by p38 mitogen-activated protein (MAP) kinase

Post-transcriptional regulation of gene expression by mitogen-activated protein kinase p38

Inhibition of SAPK2a/p38 prevents hnRNP A0 phosphorylation by MAPKAP-K2 and its interaction with cytokine mRNAs

The involvement of AU-rich element-binding proteins in p38 mitogen-activated protein kinase pathway-mediated mRNA stabilisation

The Mnks are novel components in the control of TNF alpha biosynthesis and phosphorylate and regulate hnRNP A1

Mitogen-activated protein kinase stabilizes mRNAs that contain cyclooxygenase-2 and tumor necrosis factor AU-rich elements by inhibiting deadenylation

The role of mitogen-activated protein kinase phosphatase-1 in the response of alveolar macrophages to lipopolysaccharide: attenuation of proinflammatory cytokine biosynthesis via feedback control of p38

Inhibition of p38 MAPK by glucocorticoids via induction of MAPK phosphatase-1 enhances nontypeable Haemophilus influenzae-induced expression of toll-like receptor 2

The stability of tristetraprolin mRNA is regulated by mitogen-activated protein kinase p38 and by tristetraprolin itself

Cutting edge: IL-4 induces suppressor of cytokine signaling-3 expression in B cells by a mechanism dependent on activation of p38 MAPK

FR167653 reduces obliterative airway disease in rats

Inhibition of early airway neutrophilia does not affect development of airway hyperresponsiveness

Inhaled p38alpha mitogen-activated protein kinase antisense oligonucleotide attenuates asthma in mice

Interleukin-1beta decreases expression of the epithelial sodium channel alpha-subunit in alveolar epithelial cells via a p38 MAPK-dependent signaling pathway

Haemophilus influenzae from patients with chronic obstructive pulmonary disease exacerbation induce more inflammation than colonizers

Inhibition of p38 mitogen activated protein kinase controls airway inflammation in cystic fibrosis

In vivo role of p38 mitogen-activated protein kinase in mediating the anti-inflammatory effects of CpG oligodeoxynucleotide in murine asthma

MAP kinase activation and apoptosis in lung tissues from patients with idiopathic pulmonary fibrosis

Acknowledgements R.N. is a Canadian Institutes of Health Research (CIHR) New Investigator and Alberta Heritage Foundation for Medical Research (AHRMR) Scholar and holds start up and operating grants AHRMR and CIHR respectively.