This article was published in an Elsevier journal. The attached copy is furnished to the author for non-commercial research and education use, including for instruction at the author’s institution, sharing with colleagues and providing to institution administration. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy Available online at www.sciencedirect.com Targeting p38-MAPK in the ischaemic heart: kill or cure? Rekha Bassi, Richard Heads, Michael S Marber and James E Clark The p38-MAPK pathway plays an important role in myocardial ischaemia/reperfusion injury and has been implicated in the regulation of cardiac gene expression, myocyte hypertrophy, inflammation, energetic metabolism, contractility, proliferation and apoptosis. The activation of p38-MAPK by dual phosphorylation during myocardial ischaemia aggravates lethal injury. However, under other circumstances activation can protect the heart, and recent evidence suggests that the mechanism of p38-MAPK activation may differ by circumstance. Determining the precise mechanism of activation during myocardial ischaemia is of considerable importance, since it may allow prevention of the detrimental, but not the beneficial, activation of p38-MAPK and lead to the identification of the relevant signalling molecules to be targeted for pharmaceutical intervention. termed ‘reperfusion injury’. Ischaemia/reperfusion injury may lead to further myocardial infarction, cardiac arrhythmias and contractile dysfunction [4]. The resultant myocardial fragility can result in left ventricular (LV) remodelling [5]. This is characterised by thinning of the infarcted myocardium, LV chamber dilation, fibrosis and hypertrophy of viable myocytes [6]. Early remodelling may be adaptive and maintain LV function, but longterm remodelling may contribute to functional breakdown and eventually pump failure [7]. Understanding the intracellular processes leading to cell death following ischaemia and unravelling the cellular and molecular mechanisms that trigger LV remodelling following MI is an invaluable approach in the development of therapies to prevent the progression of heart failure. Address King College London, Department of Cardiology, Cardiovascular Division. Rayne Institute, St Thomas’ Hospital, London SE1 7EH, UK p38-MAPK Corresponding author: Clark, James E (james.2.clark@kcl.ac.uk) Current Opinion in Pharmacology 2008, 8:141–146 This review comes from a themed issue on Cardiovascular and renal Edited by Antoine Bril and Metin Avkiran Available online 5th March 2008 1471-4892/$ – see front matter # 2008 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coph.2008.01.002 Introduction Ischaemic heart disease is a major cause of morbidity and mortality, accounting for 20% of all deaths in the UK [1]. Although effective interventional therapies have been devised to treat acute coronary syndromes, atherosclerotic plaques within coronary arteries still cause rapid occlusion, myocardial infarction (MI) and death [2]. MI generally results from plaque rupture/erosion with secondary thrombus formation in a coronary artery, culminating in an acute reduction of blood supply to the myocardium and is characterised by the rapid development of myocardial necrosis [3]. MI affects cardiac performance through loss of functional myocardium and total occlusion of the vessel for as little as 20–30 min can result in irreversible myocardial injury. Reperfusion is the definitive treatment for acute coronary syndromes, especially acute myocardial infarction; however, reperfusion has the potential to exacerbate lethal tissue injury, a process www.sciencedirect.com p38-Mitogen-activated protein kinase (p38-MAPK) has emerged as an important mediator of ischaemic injury, as well as a variety of other biological processes including inflammation, cell growth, cell differentiation, cell cycle and cell death in many tissue types [8]. In the heart, the p38-MAPK pathway has been implicated in the regulation of cardiac gene expression, myocyte hypertrophy, inflammation, energetic metabolism, contractility, proliferation and apoptosis [9–14]. It was first described as a 38 kDa protein that became rapidly tyrosine phosphorylated in cells stimulated with bacterial lipopolysacharride [15]. Molecular cloning revealed it to be a member of the highly conserved MAPK subfamily and was found to be the target of pyridynylimadazole compounds that blocked production of pro-inflammatory cytotokines [16]. Four isoforms of p38-MAPK, a, b, g and d, have been identified that share structural homology but differ in their sensitivity to pyridynylimadazole inhibitors (such as SB203580). p38a-MAPK and b are expressed ubiquitously in many tissues and are equally sensitive to SB203580, whereas p38g-MAPK (expression is restricted to muscle) and p38d-MAPK (predominantly found in the lungs and glomeruli) are resistant to SB203580 inhibition owing to structural differences in the ATP binding pocket [17,18]. Activation of p38-MAPK p38-MAPK is composed of two domains: a 135-residue Nterminal domain composed largely of b-sheets and a 225residue C-terminal domain being largely helical and containing the catalytic site, magnesium binding sites, and phosphorylation lip [17]. The catalytic (ATP binding) site lies at the junction between the two domains [19], and the common docking domain is located towards the C-terminus and is involved in the specific binding of Current Opinion in Pharmacology 2008, 8:141–146 Author's personal copy 142 Cardiovascular and renal upstream activators, substrates and phosphatases to p38MAPK [20]. The p38-MAPK phosphorylation lip, which corresponds to residues Gly170-Thr185 is phosphorylated on threonine (Thr180) and tyrosine (Tyr182) during classical activation. When inactive, the catalytic site is twisted so that ATP cannot bind and the phosphorylation lip is thought to occupy the peptide binding channel that lies at the interface of the amino and carboxyl-terminal domains. Upon phosphorylation, the activation loop refolds and is released from the peptide binding channel, resulting in rotation of the amino and carboxyl-terminal domains. This conformational change allows interaction between Lys53 and Asp168, which are essential for kinase activity in the catalytic site to enable ATP to bind [17,21]. p38-MAPK resides in both the nucleus and cytoplasm of resting cells, but upon activation, it can translocate to the nucleus [22]. Activators of p38-MAPK include mitogenactivated protein kinase kinase kinase 3 (MKK3), which appears to favour phosphorylation of p38a and p38b isoforms, and MKK6, which phosphorylates all p38MAPK isoforms. MKK4, originally described as a Jun N-terminal kinase activator, has also been shown to activate p38a- and p38d-MAPK and MKK7 has been reported to activate p38d-MAPK [23–25]. MKKs are themselves activated by phosphorylation on Ser/Thr residues by numerous MAP kinase kinase kinase (MAPKKKs), which are part of the response cascade to various physical and chemical stresses, such as oxidative stress, heat shock, UV irradiation, hypoxia, ischaemia, and exposure to pro-inflammatory cytokines (IL-1 and TNF) [26,27]. Mitogen-activated protein kinase-activated protein kinase 2 (MK2) was the first identified substrate of p38a-MAPK and has been extensively studied. Once activated, it phosphorylates various substrates including heat shock protein 27 (HSP27) [28], lymphocyte-specific protein 1 (LSP1) [29] and cAMP response element binding protein (CREB) [30]. A broad range of transcription factors are phosphorylated by p38-MAPK, which include activating transcription factor 1, 2 and 6, ARF accessory protein (Sap1), The CAAT/enhancer binding protein homologous transcription factor CHOP, myocyte enhancer factor 2C (MEF2C), E-26 like protein 1 (ELK1), nuclear factor of activated T cells (NFAT) and high mobility group-box protein 1 (HBP1) [31]. p38-MAPK in the heart: kill or cure? Many reports demonstrate that during myocardial ischaemia p38-MAPK activation enhances lethal injury [32–34] and inhibition protects against it [32,35–38]. However, there is also evidence to suggest that p38-MAPK activation confers protection to the heart [39–44], much of which is from investigators studying the phenomenon of ischaemic preconditioning (IPC). IPC, a powerful adaptive mechanism, was initially described in 1996 as a mechanism whereby a short duration of ischaemia protects the myocardium against a later lethal ischaemic Current Opinion in Pharmacology 2008, 8:141–146 stress [45]. IPC has been shown to activate p38-MAPK in isolated perfused rat hearts [41,42], and can be eliminated in the rat heart by treatment with SB203580, suggesting that p38-MAPK activation contributes to its protective effects. Using p38-MAPK phosphorylation as a readout of activation, Mocanu et al. demonstrated that p38-MAPK activity during ischaemia was raised above that of controls following IPC and, since cardio-protection correlates with higher levels of p38-MAPK activity during sustained ischaemia, suggested that p38-MAPK plays a crucial role in IPC [43]. The data imply that p38-MAPK activation can be both protective as well as detrimental; kill and cure? Of the four isoforms, p38a-MAPK and b are prevalent in the heart, and are similar in structure but have important functional differences, which might explain this dilemma. The first evidence for the divergent functions of p38aand p38b-MAPK was provided by Wang et al.: Using adenoviral-mediated co-expression of p38a- and p38bMAPK in neonatal rat cardiac myocytes, they demonstrated that p38a-MAPK has pro-apoptotic effects, whereas overexpression of the b isoform results in a hypertrophic phenotype [46]. Subsequently, we and others have reported a protective role for p38b-MAPK [35,47,48,49]. Using adenoviral-mediated overexpression of p38a- and p38b-MAPK, we reported that p38aMAPK is activated and p38b-MAPK deactivated following 2.5 h of simulated ischaemia. Moreover inhibition of the a isoform, but not b, led to an increase in cell viability and protection [35]. Further evidence of the opposing effects of p38a- and p38b-MAPK was provided by Kim et al. when they reported that inhibition of the a isoform following activation by reactive oxygen species significantly prevented cell death and was associated with augmented p38b-MAPK activity [50]. Importantly, none of these studies involve manipulation of endogenous p38b-MAPK; they instead rely on association or ectopic expression. Nonetheless, the evidence presented to date certainly supports the concept that IPC is the result of selective activation of p38b-MAPK. This perhaps explains why pharmacological inhibition of p38-MAPKs a and b during preconditioning blocks protection (since b is the dominant form activated), while during lethal ischaemia, the same inhibitors at identical concentrations cause protection (when the a isoform is activated). We have demonstrated the detrimental effect of p38a-MAPK activation during ischaemia, since the protective effect of SB203580 in isolated cardiomyocytes exposed to simulated ischaemia is lost when transfected with a form of p38a-MAPK resistant to pharmacological inhibition [38]. Similarly, mice heterozygous for a p38a-MAPK null allele, with reduced levels of myocardial p38a-MAPK, are resistant to infarction [51]. The evidence presented to date certainly supports the concept that the outcome of p38-MAPK activation is dependant upon the isoform that is affected and it is possible, therefore, that p38-MAPK www.sciencedirect.com Author's personal copy Targeting p38-MAPK in the ischaemic heart: kill or cure? Bassi et al. 143 isoforms are differentially activated by IPC [35]. Dissecting the role of each isoform is not easy since the IC50 values of SB-like compounds for the two isoforms are very similar [52] and selective inhibition of each isoform is not possible. More importantly, SB compounds are not wholly selective for p38-MAPK. PI3-Kinase [53], C-Jun N-terminal kinase 2 (JNK2) [54], Cyclooxygenase (COX) 1 and 2 [55], Raf kinase[56] are reportedly targeted by this class of inhibitors and, more recently, protein kinase receptor interacting protein-2 (RIP2), which is activated by a number of stimuli that are potent activators of p38MAPK, such as bacterial cell wall [57]. Therefore, the use of existing inhibitors to define the role of p38-MAPK in physiological responses may not be the correct approach. Targeted disruption of the mouse p38a-MAPK gene results in embryonic lethality owing to severe defects in placental development [58,59]. Mice lacking p38b-, p38g- or p38d-MAPK are viable and are potentially useful tools in dissecting out which isoform(s) of p38-MAPK is responsible for the effect of p38-MAPK activation in ischaemia/reperfusion injury, in a more targeted approach. To overcome the lethality of homozygous p38a-MAPK / mice, transgenic mice expressing dominant negative form of the kinase (p38a-MAPKdn) or with tissue-specific depletion of p38a-MAPK have been generated. Kaiser et al. demonstrated that transgenic mice overexpressing p38a-MAPKdn were significantly protected from myocardial ischaemia-reperfusion injury [60]. Although targeted mouse lines can help to determine the contribution of each isoform to injury, in order to understand the role of p38-MAPK in myocardial ischaemia/reperfusion a better understanding of p38-MAPK regulation is required. The duration and magnitude of p38-MAPK activation are determined by the competing activities of upstream MKKs and protein phosphatases. A family of dual-specificity MAPK phosphatases (MKPs) counteract the activity of p38-MAPK by de-phosphorylating Thr180 or Tyr182 residues in the activation lip [61,62]. At least 10 mammalian dual-specificity phosphatases have been identified. MKP-1, MKP-4 and MKP-5 have been shown to efficiently de-phosphorylate p38a- and p38b-MAPK by means of in vitro and transient transfection studies. However, p38g-MAPK and p38d-MAPK are resistant to all MKP family members. The activity of MKPs is under tight transcriptional control, and they seem to display specific tissue, cell and subcellular distributions [63]. Studies in yeast have revealed that other types of phosphatase such as protein tyrosine phosphatase (PTPase) and a serine/threonine protein phosphatase type 2C (PP2C) have important roles in downregulating the p38-MAPK pathway. Mammalian PP2Ca has been reported to negatively regulate the human MKK6 and MKK4, in vitro and in vivo. Consequently, different www.sciencedirect.com phosphatases function at different levels to inactivate p38-MAPK [25]. The traditional view of p38-MAPK activation by phosphorylation of the Thr180-Gly181-Tyr182 motif in the phosphorylation lip by MKK3 and MKK6 has been challenged by the emergence of a number of MKK-independent activation mechanisms for p38-MAPK. For example, p38a-MAPK activation during ischaemia does not involve MKKs but occurs by association of p38-MAPK with the scaffold protein TAB1 [64–66]. Another MKK-independent activation pathway involves T cell receptor induced activation through zeta-chain-associated protein kinase 70 (ZAP70) [67], whereby p38-MAPK becomes phosphorylated on Tyr323, which leads to ‘autophosphorylation’ on residues Thr180 and Tyr182. Not surprisingly, activation of p38-MAPK in response to stimuli is not limited to a single pathway. In a recent article, Kang et al. elegantly demonstrated that more than one p38aMAPK activation pathway could be activated in the same cell line by a single stimulus [68]. Using embryonic fibroblasts from MKK3/6 and MKK4/7-targeted mice, they demonstrated that p38a-MAPK activation occurred in a TAB1-dependent manner and that the presence of TAB1 in the complex had an inhibitory effect on the formation of an 85-kDa disulfide complex with an unknown binding partner in response to peroxynitrite [68]. This clearly demonstrated that p38a-MAPK activation mechanisms could be both cell type and stimulus dependent. Current research is not restricted to finding novel forms of p38-MAPK activation; a recent report by Peregrin et al. has described a novel mechanism for inactivating p38MAPK[69]. GRK2, a member of the highly conserved Gprotein-coupled receptor kinase family, can associate with p38-MAPK and phosphorylate it at Thr123, a residue located within the docking groove. This phosphorylation interferes with p38-MAPK binding affinity to MKK6 in vitro and in cells and with association and activity towards different substrates in vitro [69]. In the context of the failing heart, GRK2 levels and activity are increased in this condition and in other cardiac pathologies [70]. The development of selective and safe inhibitors of the p38-MAPK pathway is ongoing and, despite the abundance of studies using p38-MAPK inhibitors in vivo, few have advanced past phase II clinical trials (reviewed in reference [71]), owing to adverse side effects such as liver toxicity [72]. Because of this and the involvement of p38-MAPK isoforms in innumerable biological processes, targeting p38-MAPK using ‘blanket’ pharmacological inhibitors is not the best approach. Paradoxically, direct inhibition of the p38-MAPK signalling cascade may not necessarily be best achieved by inhibiting p38-MAPK itself; targets upstream or downstream could also be selected once the detrimental triggers or substrates of Current Opinion in Pharmacology 2008, 8:141–146 Author's personal copy 144 Cardiovascular and renal MI-induced p38-MAPK activation have been better characterised. Here, we have highlighted how p38-MAPK activation can be regulated by multiple mechanisms, and that there are different modes of activation in response to various stimuli in diverse cell types. The complexity in temporal profiles of p38-MAPK activation, the existence of positive and negative feedback loops, intracellular trafficking and composition of signalling complexes under different conditions add complexity and uncertainty to the precise role of p38-MAPK in myocardial ischaemia/reperfusion injury. 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Tanno M, Bassi R, Gorog DA, Saurin AT, Jiang J, Heads RJ, Martin JL, Davis RJ, Flavell RA, Marber MS: Diverse mechanisms of myocardial p38 mitogen-activated protein kinase activation: evidence for MKK-independent activation by a TAB1-associated mechanism contributing to injury during myocardial ischemia. Circ Res 2003, 93:254-261. 67. Salvador JM, Mittelstadt PR, Guszczynski T, Copeland TD, Yamaguchi H, Appella E, Fornace AJ Jr, Ashwell JD: Alternative p38 activation pathway mediated by T cell receptor-proximal tyrosine kinases. Nat Immunol 2005, 6:390-395. 68. Kang YJ, Seit-Nebi A, Davis RJ, Han J: Multiple activation  mechanisms of p38alpha mitogen-activated protein kinase. J Biol Chem 2006, 281:26225-26234. This extensive study provides evidence of multiple mechanisms of p38a activation that exist that can be used by cells to differentially respond to a variety of physiological and pathological stimuli. 69. Peregrin S, Jurado-Pueyo M, Campos PM, Sanz-Moreno V, Ruiz-Gomez A, Crespo P, Mayor F Jr, Murga C: Phosphorylation Current Opinion in Pharmacology 2008, 8:141–146 of p38 by GRK2 at the docking groove unveils a novel mechanism for inactivating p38MAPK. Curr Biol 2006, 16:2042-2047. 70. Penela P, Murga C, Ribas C, Tutor AS, Peregrin S, Mayor F Jr: Mechanisms of regulation of G protein-coupled receptor kinases (GRKs) and cardiovascular disease. Cardiovasc Res 2006, 69:46-56. 71. Clark JE, Sarafraz N, Marber MS: Potential of p38-MAPK  inhibitors in the treatment of ischaemic heart disease. Pharmacol Ther 2007, 116:192-206. An extensive review, detailing the mechanisms, circumstances and consequences of p38 activation in the heart and evaluation of p38 inhibition as a potential therapy for ischaemic heart disease. 72. Lee MR, Dominguez C: MAP kinase p38 inhibitors: clinical  results and an intimate look at their interactions with p38alpha protein. Curr Med Chem 2005, 12:2979-2994. An excellent review of recent, reported clinical results of p38 inhibitors with detailed accounts of the binding modes of these inhibitors. www.sciencedirect.com