"Stress-Responsive" Mitogen-Activated Protein Kinases (c-Jun N-Terminal Kinases and p38 Mitogen-Activated Protein Kinases) in the Myocardium

From the NHLI Division (P.H.S.) and Division of Biomedical Sciences (A.C.), Imperial College School of Medicine, London, UK.

Correspondence to Peter H. Sugden, D Phil, NHLI Division (Cardiac Medicine), Imperial College School of Medicine, Dovehouse Street, London SW3 6LY, UK. E-mail p.sugden{at}ic.ac.uk

Key Words: cardioprotection • mitogen-activated protein kinase • cellular stress • G protein–coupled receptor • hypertrophy/apoptosis

Mitogen-Activated Protein Kinase Cascades: Terminology and Properties

The best-characterized subfamilies of the mitogen-activated protein kinase (MAPK) superfamily are the extracellularly responsive kinases (ERKs) and the two "stress-responsive" MAPK subfamilies, namely, the c-Jun N-terminal kinases (JNKs) and the p38-MAPKs.^{1 2 3 4 5} As yet, no single nomenclature has been determined, and the synonyms currently in use are summarized in Table 1. The ERK cascade is the most thoroughly studied of the MAPK cascades, and it is activated principally by G protein–coupled receptor (GPCR) agonists in cardiac myocytes. We have reviewed this topic recently,⁶ and we will not discuss it in any depth here. The regulation of the JNK and p38-MAPK cascades in the myocardium (Figure 1) forms the principal subject of this review.

View larger version (24K): [in this window] [in a new window]   Figure 1. A scheme for the activation of stress-responsive MAPK cascades. Exposure of cultured cardiac myocytes and whole hearts to stresses or GPCR agonists leads to activation of the JNKs and p38-MAPKs. Cellular stresses induce reorganization of the cytoskeleton and activation of the small G proteins, Rac and Cdc42. GPCR agonists activate the small G protein, Ras, through a PKC-dependent mechanism. Ras is recognized to participate in the activation of the ERK cascade and, either directly or indirectly, may activate the stress-responsive MAPKs (JNKs and p38-MAPKs). The JNKs are activated by MKK4 and MKK7, whereas p38-MAPKs are activated by MKK3 and MKK6 (see Table 2). The upstream activators (the MKKKs) of the stress-responsive MKKs have not been clearly defined but possibly include MEKKs (MAPK or ERK kinase kinases), mixed lineage kinases (MLKs), and/or p21-activated kinases (PAKs). Substrates for the stress-responsive MAPKs include transcription factors, which regulate the transcriptional changes, and MAPKAPK2, which phosphorylates Hsp25/27 and may thereby confer cytoprotection. CHOP indicates C/EBP homologous protein; MEF2C myocyte enhancer factor 2C.

An ever-increasing number of isoforms of MAPKs are being characterized, displaying varying degrees of homology to one another. At least ten JNKs, derived from alternative splicing of three genes, have been identified.⁷ The predicted molecular masses of these isoforms are 46 or 54 kDa, depending on the absence or presence of a C-terminal extension, and activities migrating in these positions on SDS-PAGE are clearly detectable in adult rat hearts and in primary cultures of cardiac myocytes prepared from neonatal rat ventricles.^{8 9} It is not clear which of the individual isoforms are present in the myocardium, although a JNK1 antibody immunoprecipitates almost all of the 46-kDa activity and a proportion of the 54-kDa activity.¹⁰ Six p38-MAPK isoforms have been cloned: the alternatively spliced p38-MAPK(₁/₂)¹¹ and p38-MAPKß₁/ß₂¹² isoforms, p38-MAPK,^{13 14} and p38-MAPK^{15 16} . The levels of p38-MAPK and p38-MAPK transcripts in human heart cDNA libraries are low compared with those of p38-MAPK() and p38-MAPKß,¹⁶ but it is not yet clear whether these patterns are reflected in the abundances of the proteins.

MAPKs are the final components of three-membered protein kinase cascades (Figure 1). They are activated by the dual phosphorylation of a Thr-Xaa-Tyr motif (to PThr-Xaa-PTyr) catalyzed by dual-specificity MAPK kinases (MKKs), and membership of a given MAPK subfamily can be assigned on the basis of the identity of the Xaa residue (Table 1). Several stress-responsive MKKs (Table 2 and Figure 1) that show some selectivity for specific MAPKs have been identified.^{3 5} The novel MKK, MKK7,¹⁷ is selective for the JNKs, whereas MKK3 and MKK6 activate the p38-MAPKs. MKK4 was first identified as an activator of JNKs but will also stimulate p38-MAPKs. The stress-responsive MKKs are themselves probably phosphorylated and activated by MKK kinases (Figure 1). The MKK kinases (MKKKs) for the JNK and p38-MAPK cascades have not been fully characterized but may include minimally four (or five)¹⁸ MEKKs (for MEK [or ERK] kinase) and other protein kinases.¹⁹ The mechanism of activation of these MKKKs is not clear. As in the ERK cascade, small G proteins of the Ras superfamily (Ras, Rac1, and Cdc42) have been implicated. These may activate protein kinases such as p21-activated kinases and mixed-lineage kinases,^{3 19 20} which may then activate the JNK and p38-MAPK cascades.

Substrates and Inhibitors of Stress-Responsive MAPKs

All MAPKs phosphorylate Ser-/Thr- residues in proteins within a (Ser-/Thr-)Pro consensus motif, but additional factors govern the precise substrate specificity. Substrates of the JNKs and p38-MAPKs include the bZip transcription factors, c-Jun and ATF2, and the ternary complex factor transcription factors, Elk1. All are phosphorylated in their transactivation domains to increase their transactivating activities. c-Jun and ATF2 form heterodimers that transactivate at cAMP response element (CRE)-like consensus sequences within a variety of gene promoter regions (including that for c-jun itself). Elk1 upregulates c-fos expression in conjunction with the serum response factor acting at the serum response element consensus sequence, although phosphorylation of neither this transcription factor nor related ternary complex factors, such as Sap1a, has yet been studied in detail in the heart. The CRE-like and serum response element consensus elements may be involved in the upregulation of c-jun and c-fos expression seen in cultured cardiac myocytes under a variety of conditions (eg, the responses to hypertrophic agonists, stretch, and metabolic and ischemia/reperfusion stresses).^{21 22 23 24 25} c-Jun also forms heterodimers with c-Fos to transactivate at activator protein-1 (AP-1)–like sites,²⁶ which are present in many gene promoter regions.

Although ERKs were shown originally to phosphorylate c-Jun in vitro at two sites (Ser-63 and Ser-73) in the N-terminal transactivation domain, it subsequently became clear that c-Jun is preferentially phosphorylated at these sites by JNKs.²⁶ These kinases are probably exclusively responsible for the phosphorylation of c-Jun in vivo. Two other sites in the N-terminal region of c-Jun (Thr-91 and Thr-93) are also phosphorylated by the JNKs, but the role of these phosphorylations is obscure.²⁷ ATF2 represents a second transcription factor phosphorylated by JNKs and p38-MAPKs. It contains three phosphorylation sites within its transactivation domain (Thr-69, Thr-71, and Ser-90),²⁸ although Ser-90 is not directly involved in transactivation.²⁸ ATF2 may be preferentially phosphorylated by p38-MAPK and/or p38-MAPK in vitro,^{12 15} and there is evidence that the three sites may be differentially phosphorylated by the p38-MAPKs.²⁹ The kinases involved in ATF2 phosphorylation in vivo have yet to be clearly identified,⁵ but both c-Jun and ATF2 are clearly phosphorylated in cultured cardiac myocytes in response to stresses.³⁰ p38-MAPK also phosphorylates the transcription factors MEF2C and CHOP10/GADD153.⁵ The regulation of these transcription factors has not yet been studied in cardiac myocytes.

Although the only substrates of JNKs so far identified are transcription factors, p38-MAPKs phosphorylate and activate other protein kinases. These include Mnk1 and Mnk2, which may regulate the activity of the translational initiation factor eIF4E.^{31 32} p38-MAPK() and p38-MAPKß also phosphorylate two homologous protein kinases, MAPK-activated protein kinases 2 and 3 (MAPKAPK2 and MAPKAPK3).¹² The presence and activation of MAPKAPK2 has been clearly demonstrated in the heart.^{9 33 34} MAPKAPK2 and MAPKAPK3 have overlapping substrate specificities, and both phosphorylate the small heat-shock protein Hsp25/27³⁵ to increase its cytoprotective activity, an action that involves stabilization of the actin cytoskeleton.³⁶ MAPKAPK2 may also directly regulate the activity of transcription factors (eg, CRE binding protein).⁵

Two relatively specific chemical inhibitors of p38-MAPK() and p38-MAPKß, SB203580 and SB202190,^{11 12 15 29 37} have been identified, and these are being increasingly used to identify biological substrates of these p38-MAPKs.⁵ Caution should be exercised because at the concentrations normally used (10 µmol/L), SB203580 inhibits recombinant JNK2 in vitro³⁸ and at least two JNK isoforms in the heart.³⁹ Examining the concentration dependence of inhibition can assist in establishing the involvement of p38-MAPKs, since inhibition should be clearly demonstrable at <1 µmol/L. There are as yet no chemical inhibitors that are selective for the JNKs. However, expression plasmids encoding the JNK interacting protein, which inhibits the stimulation of gene expression by activated JNKs,⁴⁰ may prove useful in transfection experiments.

Activation of JNKs and p38-MAPKs in the Myocardium by Cellular Stresses

As might be predicted from studies in other systems, cellular stresses such as hyperosmotic shock, low concentrations of protein synthesis inhibitors (eg, anisomycin), hypoxia/reoxygenation, and reactive oxygen species (ROS) activate the JNKs in cultured cardiac myocytes.^{8 41 42 43 44} The JNKs are also activated by the proinflammatory cytokines, interleukin-1ß and tumor necrosis factor-.⁴¹ The activation of p38-MAPKs in myocytes has not been as fully characterized, but these MAPKs are activated by the cellular stresses that have been examined (ROS,^{44 45} hypoxia/reoxygenation,⁴³ hyperosmotic shock,⁴⁶ arsenite,⁴⁷ and proinflammatory cytokines; authors' unpublished data, 1998). Activation of p38-MAPK by cellular stresses is associated with activation of MAPKAPK2 and the phosphorylation and disaggregation of Hsp25/27.⁴⁴ There is minimal information on the upstream mechanisms of activation of the JNKs and p38-MAPKs in myocytes, although PAK is activated by hyperosmotic stress and hypoxia/reoxygenation but not by interleukin-1ß or endothelin-1 (ET-1).^{43 48}

Probably the most pathologically relevant forms of cardiac stress in vivo are ischemia and ischemia/reperfusion, and it is clear that these stresses powerfully activate the stress-responsive MAPKs in the intact isolated rat heart. p38-MAPK(s) and MAPKAPK2 are activated during ischemia, and their activation is sustained or increased during reperfusion.^{9 25} The activation of MAPKAPK2 is completely inhibited by SB203580, implicating particularly p38-MAPK() and/or p38-MAPKß in its activation in the heart.¹⁰ In contrast, the JNKs are not activated during global ischemia but are strongly activated during the reperfusion phase.^{9 25 49} This differential activation of the JNKs and p38-MAPKs is unexpected, because in most systems the two pathways tend to be activated in parallel.

As mentioned above, JNKs and p38-MAPKs phosphorylate c-Jun and ATF2 to increase the transactivating activity of c-Jun/ATF2 heterodimers, and both are phosphorylated in cultured cardiac myocytes subjected to cellular stresses.³⁰ The c-jun promoter contains two CRE-like sequences that bind c-Jun/ATF2. The promoter for ATF3, the gene encoding another transcription factor, contains one such site. After ligation of the left anterior descending coronary artery, c-jun and ATF3 expression is increased in the ischemic zone.²⁵ On reperfusion, the expression becomes ubiquitous.²⁵ It is probable that phosphorylation and activation of c-Jun/ATF2 by JNKs and p38-MAPKs is involved in the upregulation of these genes after ischemia and ischemia/reperfusion.

What might be the mediators for the activation of JNKs and p38-MAPKs during ischemia and/or ischemia/reperfusion in the isolated heart? Numerous biochemical changes occur within the heart under these conditions, including increased oxidative stress and production of ROS, changes in ion (eg, H⁺ and Ca²⁺) homeostasis and energy/fuel metabolism, decreases in intracellular concentrations of ATP and creatine phosphate, degradation of adenine nucleotides to adenosine and other nucleosides, and osmotic disturbances. Oxidative stress, as exemplified by low concentrations of H₂O₂, activates JNKs, p38-MAPK, and MAPKAPK2 in perfused hearts.¹⁰ Although the particular species may differ, many studies have shown that ROS are formed during ischemia and on subsequent reperfusion (eg, see References 50 and 51^{50 51} ). For example, in chick embryo cardiac myocytes, superoxide anion and H₂O₂ are produced during simulated ischemia, whereas OH· and H₂O₂ are produced during simulated reperfusion.⁵¹ The formation of any single ROS will generate other species (eg, OH· from H₂O₂ by the Fenton reaction). The lipophilic spin trap N-tert-butyl--phenyl nitrone prevents activation of p38-MAPK during ischemia, whereas DMSO (an OH· scavenger) diminishes the activation of JNKs and p38-MAPKs in reperfused hearts, suggesting that the various ROS may differentially activate the stress-responsive MAPKs.¹⁰ Similarly, antioxidants inhibit the activation of JNKs in cultured cardiac myocytes subjected to hypoxia/reoxygenation⁴² ; conversely, depletion of intracellular glutathione increases the activation of JNKs in cells subjected to oxidative stress.⁴² These findings suggest that ROS are at least in part responsible for the activation of JNKs and p38-MAPKs during ischemia and ischemia/reperfusion. The situation is more complex in vivo, where an inflammatory response is likely to be triggered stimulating the release of proinflammatory cytokines, which may also activate cardiac stress-responsive MAPKs.

Activation of JNKs and p38-MAPKs in the Myocardium by GPCR Agonists

GPCR agonists such as ET-1,⁸ the -adrenergic agonist phenylephrine (PE),^{8 52} and angiotensin II (Ang II)⁵³ activate JNKs in cultured cardiac myocytes, although this activation is considerably less than that obtained with cellular stresses (such as hyperosmotic shock or the protein synthesis inhibitor, anisomycin).⁸ Likewise, p38-MAPK and MAPKAPK2 are activated by ET-1 and PE, and this leads to phosphorylation of Hsp25/27.⁴⁶ ET-1 and PE stimulate the phosphorylation of c-Jun and ATF2 in cultured cardiac myocytes,⁵⁴ presumably through the JNKs and p38-MAPKs. In the intact perfused rat heart, the JNKs, p38-MAPK, and MAPKAPK2 are activated by PE.⁵⁵ Mechanical stresses (passive stretch⁵⁶ and electrical pacing⁵⁷ ) activate JNKs in cultured cardiac myocytes. The effects of stretch on p38-MAPK and MAPKAPK2 activities in cardiac myocytes have not yet been studied, but increasing wall stress in the intact heart by perfusion at "hypertensive" pressure activates p38-MAPK, MAPKAPK2, and the JNKs.¹⁰ There is evidence that stretch causes the release of Ang II and/or ET-1 from cells,^{58 59} and these may have an autocrine/paracrine effect to stimulate JNKs and p38-MAPKs.

An important signaling pathway for ET-1, PE, and Ang II is their G_qPCR-mediated stimulation of phosphoinositide hydrolysis and activation of the diacylglycerol-regulated isoforms of protein kinase C (PKC). The pharmacological activator of the diacylglycerol-regulated PKCs, phorbol 12-myristate 13-acetate (PMA), is only a weak activator of JNKs and p38-MAPK in cultured cardiac myocytes.^{8 46} However, PMA synergizes with the Ca²⁺ ionophore A23187 to increase JNK activity, and chelation of intracellular Ca²⁺ inhibits JNK activation by Ang II.⁵³ Downregulation of diacylglycerol-regulated PKC isoforms inhibits activation of JNKs by Ang II⁵³ and p38-MAPK/MAPKAPK2 by ET-1.⁴⁶ The PKC-selective inhibitor, GF109203X, also inhibits activation of p38-MAPK and MAPKAPK2 by ET-1.⁴⁶ These data implicate PKC in the activation of JNKs and p38-MAPK by GPCR agonists, although they also suggest that stimulation of PKC alone is not sufficient for full activation.

Stress-Responsive MAPKs and Hypertrophy of the Cardiac Myocyte

The hypertrophic response of the myocardium is an important pathophysiological adaptation that is associated with alterations in gene expression and cell morphology (increased sarcomeric assembly and myocyte profile).²¹ ET-1, PE, and PMA are powerful hypertrophic agonists in cultured cardiac myocytes. It was suggested in 1993 that activation of the ERK cascade by these agonists may mediate the hypertrophic response.⁶⁰ Although there is considerable evidence that the ERKs participate in myocyte hypertrophy,⁶¹ it is now apparent that ERKs are not the sole mediators of this response.^{62 63 64} Indeed, some investigators believe that they may even be inhibitory.^{62 64 65} Because GPCR agonists are now known to activate JNKs and p38-MAPKs, the role(s) of these pathways in hypertrophy is under investigation.

Stress-Responsive MAPK Cascades and Hypertrophy
Transfection of neonatal cardiac myocytes with constructs for constitutively active MEKK1 (an MKKK that preferentially activates the JNK cascade) in the presence or absence of MKK4 stimulates expression of atrial natriuretic factor (ANF), ß-myosin heavy chain, and skeletal muscle -actin.^{9 52 65} This pattern of gene expression is associated with hypertrophy in the rat.²¹ MEKK1 in combination with MKK4 increases the myocyte profile but does not induce the myofibrillar organization that typifies a true hypertrophic response.⁹ Transfection of a construct for inhibitory ("dominant-negative") MEKK1 inhibits PE-induced ANF expression, suggesting that the JNK pathway may be necessary for ANF gene upregulation.^{52 65} This construct also attenuates the stimulation of c-Jun–transactivating activity by PE, establishing a potential link through MEKK1 and JNKs to transcription.⁵² These experiments are not necessarily unequivocal; although MEKK1 may activate the JNK cascade preferentially, it also activates ERKs and p38-MAPK in cultured cardiac myocytes.^{65 66} However, a dominant-negative JNK construct inhibits PE-induced ANF expression,⁶⁵ further implicating the JNK cascade in the hypertrophic response.

Recently, p38-MAPK has been proposed to effect cardiac hypertrophy. Transfection of neonatal cardiac myocytes with constitutively activated MKK3 or MKK6, both of which preferentially activate p38-MAPKs, stimulates expression of ANF and skeletal muscle -actin and increases cell profile and myofibrillar assembly.^{66 67} MKK6 increases p38-MAPK activity but does not stimulate ERKs or JNKs.⁶⁶ It was consistently more effective than activated c-Raf (which activates only the ERK cascade) or activated MEKK1 in inducing the transcriptional and morphological changes associated with hypertrophy.⁶⁶ In that study (Zechner et al⁶⁶ ), PE induced the phosphorylation of both p38-MAPKs and ERKs (but confusingly not the JNKs) and, as expected, stimulated a hypertrophic response. The hypertrophic responses induced by PE, MKK3, or MKK6 are reduced by p38-MAPK()/p38-MAPKß inhibitors.^{66 67} However, the involvement of p38-MAPKs in cardiac hypertrophy may not be simple. Constitutively activated MKK3 diminishes cell survival, and cotransfection experiments (with p38-MAPK() and p38-MAPKß constructs show that this effect is primarily dependent on p38-MAPK().⁶⁷ In contrast, the hypertrophic action of MKK3 appears to be primarily mediated by p38-MAPKß. To confuse matters, a recent study from the same group (Wang et al⁶⁸ ) shows selective activation of JNKs and induction of hypertrophy in cultured cardiac myocytes by transfected wild-type or constitutively activated MKK7. This group of investigators now appears to propose a role for JNKs in stimulating hypertrophy and a role for p38-MAPKs in promoting cell death.

Most of the data relating to the hypertrophic response have been obtained in myocytes after 48 hours of transfection or agonist stimulation. We have examined in more detail the time course of development of the morphological changes associated with PE or ET-1–induced hypertrophy in cultured cardiac myocytes.⁴⁶ Our results indicate that increases in cell profile and myofibrillar organization are apparent from as early as 4 hours of stimulation and that inhibition of p38-MAPK()/p38-MAPKß with SB203580 has no effect on these changes over the first 24 hours.⁴⁶ However, although cells stimulated with PE or ET-1 retain their hypertrophic morphology at 48 hours, cells stimulated with either agonist in the presence of SB203580 appear smaller, with no sarcomeric structure and minimal immunostaining for ß-myosin heavy chain. This suggests that rather than initiating the hypertrophic response, activation of p38-MAPK by PE or ET-1 may be more important in its maintenance over a longer period of time. Furthermore, over the 48-hour period, control cells cultured in serum-free conditions regress, becoming progressively smaller⁴⁶ and undergoing apoptosis.⁶⁹ These data suggest that studies of apparent hypertrophy after 48 hours may instead reflect cell survival.

Small G Proteins and Hypertrophy
Small G proteins (eg, Ras, Rac, and Cdc42) are active in their GTP-ligated state and are implicated upstream from the MAPK cascades. PE increases Ras.GTP loading in cultured cardiac myocytes,⁵² transfection with constitutively activated Ras induces a hypertrophic response,⁷⁰ and transgenic mice that express activated Ras in the heart develop cardiac hypertrophy.⁷¹ Ras may be involved in the activation of several signaling pathways (Figure 1). It has been known for several years that the ERK cascade is activated by Ras.GTP in many cells, including cultured cardiac myocytes.⁶⁶ Ras has more recently been implicated in the activation of the JNK cascade,¹⁹ and transfection of a construct for constitutively activated Ras activates JNKs in cultured cardiac myocytes in the hands of some investigators.⁵² However, in another study, constitutively activated Ras failed to activate either JNKs or p38-MAPKs.⁶⁶ Clarification of these discordant data is necessary.

Other small G proteins of the Rho subfamily (Rac and Cdc42) have been implicated in the activation of the stress-responsive MAPKs in many cell types.^{19 20} Transfection of cultured cardiac myocytes with constitutively activated Rac selectively increases JNK activity and has some hypertrophic effects.⁶⁶ RhoA itself has been excluded from the activation of stress-responsive MAPKs in most cell types,^{19 20} but transfection of myocytes with constitutively activated RhoA stimulates expression of ANF, and inhibition of RhoA activity prevents the hypertrophic response to PE.^{65 72 73} How RhoA relates to the various signaling pathways governing the response of myocytes to PE remains to be determined. These findings suggest that an examination of the role of various recently discovered Rho-activated protein kinases in myocyte hypertrophy might be productive.

General Overview of Stress-Responsive MAPKs and Hypertrophy
Many of the studies seeking to establish a role for MAPKs in cardiac hypertrophy have used transfection protocols, occasionally in combination with measurements of MAPK activities. The usual methodology involves transfection of a fusion gene in which the promoter region of a "marker gene" of cardiac hypertrophy (eg, ANF) is fused with a reporter construct (eg, firefly luciferase). Expression vectors encoding activated or inhibitory signaling intermediates (eg, MEKK1) are cotransfected, and the expression of luciferase activity is taken as an index of transcriptional activation. From the preceding discussion, it is clear that transfection experiments produce often divergent or contradictory data, leading to much confusion. There are many potential reasons for this. The precise methodologies used by different groups vary, and the varying degrees of overexpression of the "transfected" signaling intermediates may produce different effects. It is particularly difficult to assess the degree of overexpression at the low transfection efficiencies in experiments that have used nonviral expression vectors. Here, there is not even universal agreement concerning the necessity for or the optimum method of normalization of data. Some of these problems may be avoided by the use of adenoviral vectors where the transfection efficiency approaches 100%. However, it should be realized that the "readout" (ie, luciferase activity in this example) represents the net result of several processes (eg, transcription, processing and nuclear export of mRNA, mRNA stability, and protein synthesis and degradation), all of which may be modulated by the invoked signaling intermediate. Furthermore, the specific activities of the constitutively activated mutant signaling intermediates may differ from those of the activated wild-type proteins, and substrate specificity may be widened when kinases of MAPK cascades are overexpressed in transfection experiments. Broadened substrate specificity is a recognized problem in analogous experiments in vitro at unphysiological concentrations or ratios of enzymes and substrates. In some cases, experiments have used constructs encoding kinases rendered constitutively active by deletion of regulatory sequences (eg, MEKK1^{9 52} ). Such deletions could unavoidably remove domains that determine substrate specificity and interaction. There are also problems with the use of dominant-negative constructs and/or "selective" chemical inhibitors, since these reagents may not be as specific as is assumed. Finally, it is becoming clear from work with other systems that transcription from transiently transfected plasmids, for example, and from genomic DNA is regulated differently. This reflects the additional complexities of organization in the latter.

In studies of hypertrophy in which MAPK activation has been examined (either in response to agonists or in transfection experiments), the degree of activation of MAPKs is often ignored. For example, it is difficult to assess the significance of a 2-fold activation of JNKs by PE^{8 52} to the overall hypertrophic response, when cellular stresses (which do not stimulate hypertrophy) increase JNK activity by 20-fold.⁸ It may be argued in this instance that the additional activation of the p38-MAPK cascade by stresses leads instead to cell death, but it is apparent that GPCR agonists such as PE also activate p38-MAPKs.⁴⁶ Equally, PMA is strongly hypertrophic²¹ but only weakly activates JNKs or p38-MAPKs.^{8 46} The only real conclusion from the myriad data is that activation of any one MAPK pathway may produce a partial hypertrophic response, and it is now clear that these data must be considered within the additional context of cell survival.^{46 67 68} Finally, it is tempting to speculate that a single signaling pathway may play a dominant role in cardiac hypertrophy. Although this may indeed be the case for pharmacological stimuli that produce a prolonged powerful activation of a single MAPK pathway (eg, PMA and the ERK cascade⁷⁴ ), the in vivo response with physiological agonists is probably much more complex and probably involves signal integration from multiple pathways.

Stress-Responsive MAPKs and Apoptosis

The JNKs and p38-MAPKs have been implicated in cell death/apoptosis in other cell types, but studies of this type in the heart are still in their infancy. Proinflammatory cytokines and ROS (which activate the stress-responsive MAPKs^{10 41 45} ) induce apoptosis in cultured cardiac myocytes.^{45 75} DNA laddering (an event associated with apoptosis) can be detected in cardiac myocytes cultured in serum-free conditions⁶⁹ and in ischemic hearts that have been perfused for long periods.²⁵ Although transfection experiments have implicated p38-MAPK() in cardiac myocyte apoptosis,⁶⁷ the association between stress-responsive MAPK activation and cardiac myocyte death/apoptosis has yet to be proved, but it remains an interesting possibility.

Ischemic Preconditioning

A brief period of sublethal ischemia protects the heart against subsequent, more severe ischemia (ischemic preconditioning).^{76 77} There are two phases, an acute phase in which the heart is protected for a few hours and a so-called "second window of protection," which appears after 24 to 36 hours and may involve transcriptional upregulation. Because of its immense therapeutic potential, the signaling pathways underlying ischemic preconditioning are under intense investigation. However, most investigations have merely attempted to simulate or inhibit the preconditioning phenomenon with a battery of agonists or inhibitors. What is tantamount to an ischemic preconditioning protocol leads to activation of JNKs and p38-MAPKs in perfused hearts.^{9 25 49} We have suggested that an examination of the involvement of these kinases in ischemic preconditioning might be fruitful,⁹ particularly since activation of p38-MAPK should lead to phosphorylation of Hsp25/27 and increase its cytoprotective capacity. Such studies are under way in a number of laboratories. In a recent report, the phosphorylation of p38-MAPK was examined in rabbit hearts over a 30-minute period of ischemia that followed a 5-minute preconditioning ischemia.⁷⁸ After some manipulation, the results suggest that phosphorylation of p38-MAPK is seen during the 30-minute ischemic period only after a preceding preconditioning ischemia. In isolated rabbit myocytes, SB203580 prevents simulated "preconditioning" (assessed by the ability of myocytes to exclude trypan blue), and anisomycin, which activates stress-responsive MAPKs, can induce preconditioning.⁷⁸ Although it is still controversial,⁷⁹ a considerable body of evidence has implicated PKC in preconditioning.^{76 77} Thus, many agonists that induce preconditioning (eg, bradykinin, ₁-adrenergic agonists, ET-1, purinergic agonists, PMA, and diacylglycerol) activate PKC, and inhibitors of PKC attenuate ischemic preconditioning. Our recent data show that p38-MAPK is activated by ET-1 and PE in a PKC-dependent manner,⁴⁶ providing a possible link between PKC and p38-MAPK, a connection that could be particularly relevant to ischemic preconditioning. Equally, the involvement of PKC in G_qPCR-mediated activation of the JNKs⁵³ may be significant in this respect. In the next few years, we are likely to see considerable advances in understanding the intracellular signaling mechanisms involved in ischemic preconditioning.

General Conclusions

Much of the basic work involving the regulation of JNKs and p38-MAPKs has been carried out in noncardiomyocytic cells (eg, fibroblasts), but the area has immense potential significance in the myocardium with respect to its reactions to pathological stresses (eg, hypoxia, ischemia, reperfusion injury, hypertension, and inflammatory disease) (see Figure 2). The regulation of the stress-responsive MAPKs in the heart is currently being intensively investigated, but the biological consequences of their activation and inhibition in the heart are still unclear. It is likely that these will become clearer over the next few years and that the stress-responsive MAPK cascades may prove to be targets that are suitable for pharmacological manipulation.

View larger version (19K): [in this window] [in a new window]   Figure 2. Regulation of myocardial responses by stress-responsive MAPKs. The biological responses to activation of stress-responsive MAPKs in the myocardium are not fully understood, but their activation may be involved in a number of pathophysiological processes.

Note Added in Proof
Nemoto et al (Nemoto S, Sheng Z, Lin A. Opposing effects of Jun kinase and p38 mitogen-activated protein kinase on cardiomyocyte hypertrophy. Mol Cell Biol. 1998;18:3518–3526) have also recently found that activation of the p38-MAPK cascade stimulates ANF expression and that chemical inhibition of the cascade prevents the morphological changes induced by hypertrophic agonists at 48 hours. Somewhat surprisingly in view of the work of others,^{9 52 65 68} activation of the JNK cascade inhibits MEKK1-stimulated ANF expression.

Acknowledgments

Our work in relation to this topic was supported primarily by the British Heart Foundation and the Wellcome Trust. Because of space limitations, many of the original articles cited refer specifically to the myocardium, and we have used reviews for the more basic information where possible. We apologize to investigators whose background articles on MAPKs have not been cited.

Received March 16, 1998; accepted May 14, 1998.

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