MINIREVIEW Role of extracellular signal regulated kinases 1 and 2 in neuronal survival Michal Hetman 1,2,3 and Agata Gozdz 1 1 Kentucky Spinal Cord Research Injury Center and Departments of Neurological Surgery and 2 Pharmacology and Toxicology, University of Louisville, KY, USA; 3 Nencki Institute, Warsaw, Poland Extracellular signal regulated kinases 1 and 2 (ERK1/2) regulate cellular responses to a variety of extracellular stimuli. In the nervous system, ERK1/2 is critical for neur- onal differentiation, plasticity and may also modulate neuronal survival. In this minireview, we present evidence that supports prosurvival activity of ERK1/2 in neurons. Several reports suggest that ERK1/2 mediates neuropro- tective activity of extracellular factors, including neurotro- phins. In addition, ERK1/2 is activated by neuronal injury. In damaged cells, ERK1/2 activation may act as a defensive mechanism that helps to compensate for the deleterious effects of a damaging insult. The emerging mechanisms of ERK1/2-mediated neuroprotection may involve transcrip- tional regulation and/or direct inhibition of cell death machinery. Keywords: apoptosis; MAP-kinase; neurons; neuropro- tection; programmed cell death; signal transduction; survival. Signaling pathways and neuronal survival Death of neurons occurs during development of the central nervous system (CNS). Therefore, restriction of neuronal death by survival signaling is critical for the proper formation of the CNS [1]. In CNS pathologies including stroke, Alzheimer’s disease or CNS trauma, nerve cell death occurs as a result of cell injury [1]. Consequently, survival signaling cascades may provide targets for neuroprotective therapies against these conditions. Neuronal survival sign- aling involves several pathways including phoshpatidylo- inositol-3-kinase (PI3K), extracellular signal regulated kinase 1/2 (ERK1/2) or extracellular signal regulated kin- ase 5 (ERK5) [2]. Interestingly, while PI3K seems to be the principal transducer for prosurvival factors involved in trophic support, several reports suggest preferential ERK1/2 involvement in protection against damage-induced cell death. Therefore, manipulations of the latter pathway may be useful for neuroprotective interventions in diseases. ERK1/2 signaling ERK1,alsoknownasp44mitogenactivatedproteinkinase (MAP kinase), and ERK2 (p42 MAP kinase) are closely related protein kinases of the MAP kinase family [3]. ERK1/2 is regulated by a cascade of phosphorylations including a dual phosphorylation at Thr/Tyr residues of the ERK1/2 activation domain that is carried out by MAP kinase kinase 1/2 (MKK1/2) [3]. These dual phosphorylation events activate ERK1/2 [3]. MKK1/2 is, in turn, activated by phosphorylation catalyzed by the Raf family of protein kinases. Raf activators include small GTPases, Ras or Rap3. ERK1/2 activity is decreased by either phosphotyrosine phosphatases or dual specificity (Ser/Thr + Tyr) phospha- tases known as MAP kinase phosphatases (MKPs) [3]. ERK1/2 is considered a Ôproline directed kinaseÕ as it phosphorylates Ser or Thr residues followed by a proline. ERK1/2 targets include several transcription factors, sign- aling mediators, cytoskeletal proteins and protein kinases [3]. ERK1/2 may also engage in the regulation of cellular processes via protein–protein interactions rather than through its kinase activity. For instance, kinase dead mutant forms of ERK2 may activate MKPs or DNA topoiso- merase II [3]. It is believed that ERK1/2 interactions with its activators and/or substrates are enhanced by scaffolding proteins such as MP-1 [3]. Also, ERK1/2 signaling may be regulated by subcellular localization [3] and crosstalk to other signaling mediators including Ca 2+ ,cAMPorPI3K [2,4]. Biological processes involving ERK1/2 include stimu- lation of cell proliferation and survival, neoplastic transfor- mation, neuronal differentiation and plasticity [2,3]. Tools to study ERK1/2 signaling Several approaches have been employed to study the biological role of ERK1/2 in mammalian cells including Correspondence to Michal Hetman, Kentucky Spinal Cord Research Injury Center, University of Louisville, 511 S. Floyd St., MDR616, Louisville, KY 40292, USA. Fax: + 1 502 852 5148, Tel.: + 1 502 852 3619, E-mail: michal.hetman@louisville.edu Abbreviations: BDNF, brain derived neurotrophic factor; CNS, cen- tral nervous system; CREB, cAMP response element binding protein; ERK, extracellular signal regulated kinase; HSV2, herpes simplex virus type 2; MAP, mitogen activated protein; MKK1/2, MAP kinase kinase 1/2; MKP, MAP kinase phosphatase; NGF, nerve growth factor; NMDA, N-methyl- D -aspartate; NMDAR, NMDA receptor; P13K, phoshpatidyloinositol-3-kinase; PARP, poly-(ADP-ribose)- polymerase; TGFa, transforming growth factor a. (Received 14 February 2004, accepted 18 March 2004) Eur. J. Biochem. 271, 2050–2055 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04133.x overexpression of mutated elements of the pathway, use of pharmacological inhibitors and genetic ablation in knock- out mice [3]. Perhaps the most popular tools to manipulate ERK1/2 signaling are the dominant-negative and the constitutive-active mutants of MKK1 as well as several pharmacological inhibitors of MKK1/2. It is important to realize the limitations of these tools. First, it is possible that MKK1/2 may also have other targets than ERK1/2 [5]. Secondly, specificity of commonly used MKK1/2 drug inhibitors is not absolute. For instance, PD98059 and U0126 may inhibit signaling by ERK5 [6]. PD98059 was also shown to directly inhibit cyclooxygenases [7] and block Ca 2+ influx into isolated synaptosomes [8]. The interpret- ation of the data obtained through experimental modula- tion of ERK1/2 signaling may be hampered by the fact that inhibition of one element of the ERK1/2 signaling network may modulate other signaling units. For instance, selective inhibition of MKK1/2 increases ERK5 activity [6]. There- fore, the optimal design of experiments addressing ERK1/2 function could include application of several inhibitors and dominant-negative mutants that act at various levels of the ERK1/2 cascade as well as testing the effects of these agents on other signaling circuitries that may crosstalk to ERK1/2. ERK1/2 as a transducer of extrinsic survival signals The first reported experiments suggesting that ERK1/2 may transduce anti-apoptotic signaling in neurons were performed in neuronally differentiated PC12 cells [9]. Because nerve growth factor (NGF) activated ERK1/2 in this system, and NGF withdrawal-induced apoptosis was prevented by overexpression of constitutively active mutants of MKK1, Xia et al. proposed that trophic signaling by NGF was mediated through the ERK1/2 pathway [9]. Additional studies showed that ERK1/2 activation may also block cell death induced by trophic deprivation of retinal ganglion cells, cerebellar granule neurons or spiral ganglion neurons. In rat retinal ganglion cells or cerebellar granule neurons, protective ERK1/2 was activated by brain derived neurotrophic factor (BDNF) [10–12], or by cAMP signaling triggered by PACAP or forskolin [10,13]. In rat spiral ganglion neurons, ERK1/2 mediated the protective effect of the neuromodulator substance P [14]. However, several other studies using both pharmacological inhibitors and various mutants of the ERK1/2 pathway suggested that ERK1/2 may not be the major mediator of neuroprotection afforded by neuro- trophins, IGF-1 or membrane depolarization in trophic- deprived neurons (reviewed in [2]). Importantly, as the studies indicating a lack of ERK1/2 involvement in antiapoptotic signaling used a variety of neuronal cells, the role of ERK1/2 in protection against trophic with- drawal may appear only in some contexts. In contrast, PI3K/Akt signaling seems to be the principal mediator of both the anti-apoptotic effects of basal trophic support and neuroprotective action of agents that suppress trophic deprivation-induced cell death [2]. Although the contribution of ERK1/2 to support neur- onal survival under ÔbasalÕ culture conditions may be minimal, there is accumulating evidence that ERK1/2 may mediate the neuroprotective activity of such factors that neuroprotect against damaging insults. For instance, ERK1/2 activation by BDNF was shown to protect cultured rat cortical neurons against apoptosis induced by DNA damage [15,16]. In these studies, DNA damage was induced by genotoxic anticancer drugs, camptothecin or cisplatin (CPDD). Interestingly, cisplatin is highly neuro- toxic, which limits its use against CNS tumors [17]. BDNF protection against neuronal apoptosis induced by either of these compounds was inhibited with MKK1/2 blockers, PD98059 or SL327 (a blood–brain barrier permeable compound similar to U0126). Because neurons that over- expressed a constitutive-active form of MKK1 were protected against camptothecin or CPDD, it seems that ERK1/2 activation is both necessary and sufficient for the anti-apoptotic action of BDNF [15,16]. Similarly, Anderson & Tolkovsky showed that in cultured sympathetic neurons exposed to another DNA-damaging agent, cytosine arabi- noside, PD98059 inhibited NGF-mediated protection, implicating ERK1/2 involvement [18]. There are several reports suggesting that ERK1/2 may serve as a transducer for agents that protect from other forms of neuronal injury including excitotoxicity, calcium overload, oxidative injury, hypoxia or neurotoxic viruses. ERK1/2 is required for neuroprotection by estrogen against glutamate excitotoxicity [19]. Also, in hippocam- pal slice cultures that were challenged by an excitotoxic insult with N-methyl- D -aspartate (NMDA), the protective effects of nicotine were ERK1/2-dependent [20]. In another study, transforming growth factor a (TGFa) was shown to protect neurons from NMDA-induced death by activating astrocytic ERK1/2. In agreement with the anti-excitotoxic effects of ERK1/2, cortical neuron death by ionomycin-induced Ca 2+ overload was attenuated by BDNF, at least in part, via ERK1/2 activation [21]. ERK1/2 also seems to contribute to the neuroprotective effects of cAMP. For instance, corticotrophin releasing hormone protected hippocampal slices from excitotoxicity by cAMP-mediated activation of ERK1/2 [22]. Also, prostaglandin E1, a signaling mediator know to activate the cAMP pathway, protected rat spinal neurons against nitric oxide, acting partially through ERK1/2 [23]. Similarly, the cAMP inducer forskolin increased the survival of cultured dopaminergic neurons by reducing the sponta- neous oxidative toxicity, via stimulation of the ERK1/2 pathway [24]. ERK1/2 activation mediates the protective effects of several factors that enhance neuronal survival in hypoxia/ ischemia models. For instance, Han & Holtzman showed that in P7 rat pups, intraventrical injection of BDNF protected against hypoxic/ischemic brain injury through ERK1/2 but not PI3K [25]. Other examples of neuropro- tectants that counteract hypoxia by activating ERK1/2 include erythropoietin, fructose-1, 6-biphosphate or N-acetyl-o-methyldopamine [26–28]. Finally, TGF1b-medi- ated ERK1/2 activation inhibited staurosporine-induced cell death of cultured hippocampal neurons and was also suggested to protect in a rat stroke model [29]. An interesting example of ERK1/2-mediated neuroprotection against injury is that induced by the herpes simplex virus type 2 (HSV2). In this case, virally produced ICP10 protein activated ERK1/2 to inhibit apoptosis of infected Ó FEBS 2004 Role of ERK1/2 in neuronal survival (Eur. J. Biochem. 271) 2051 hippocampal neurons [30]. Thus, ERK1/2 appears to be required for several neuroprotectants that support neuronal survival after injury. Activation of ERK1/2 as a defense mechanism in neuronal injury Neurons respond to cell damage by activation of death signaling pathways. However, at the same time, cells may also mobilize defense mechanisms in an attempt to coun- teract cell death and enable damage repair. Interestingly, defensive ERK1/2 activation is observed after neuronal injuries. ERK1/2 activation was observed in cultured cortical or hippocampal neurons exposed to the DNA damaging agents, camptothecin, CPDD or etoposide [15,16,31]. In cultured rat cortical neurons, CPDD-mediated activation of ERK1/2 reached levels similar to those observed at the peak of BDNF action [16]. Inhibition of ERK1/2 by either pharmacological inhibitors or by a dominant-negative mutant form of MKK1 increased CPDD-induced apoptosis and further reduced survival of CPDD-treated neurons [16]. Surprisingly, the protective ERK1/2 activation turned out to be mediated by NMDA receptors (NMDARs) [16]. Further experiments indicated that in CPDD-treated neurons, NMDAR signaling to ERK1/2 is enhanced by activation of poly-(ADP-ribose)- polymerase (PARP), an enzyme that mobilizes DNA repair but may also lead to energetic deprivation and necrosis [16]. Elevated PARP activity was suggested to stimulate NMDAR signaling by depleting neurons of ATP [32]. Because the moderate increase in PARP activity observed in CPDD-treated neurons did not deplete cellular ATP, PARP contribution to the defensive ERK1/2 activation may result from a mechanism that does not involve disturbed neuronal energetics [16]. Therefore, it appears that DNA damage by CPDD may activate a novel neuronal defense circuitry that engages PARP, NMDAR and ERK1/2. It remains to be tested if a similar pathway may contribute to the defense against other genotoxic insults. The role of ERK1/2 in neuronal survival after exposure to DNA-damaging anti- cancer agents suggests that neurotoxicity induced during cancer treatment may be enhanced by drugs affecting protective ERK1/2 activation including the clinically used NMDAR antagonist, memantine. ERK1/2 seems to be an important defense mechanism against hypoxia/ischemia. Induction of ischemic tolerance by an episode of mild ischemia can be modeled in cultured rat cortical neurons by transient oxygen/glucose depriva- tion [33]. Gonzalez-Zulueta et al. showed that in this system, protective oxygen/glucose deprivation precondi- tioning activated NMDARs resulting in increased produc- tion of NO and subsequent activation of Ras, which ultimately signaled to ERK1/2 [33]. The inhibition of ERK1/2 activation in this case completely abolished the protective effects of preconditioning [33]. Furthermore, in mouse cortical neurons that were exposed to cytotoxic hypoxia, ERK1/2 was activated and ERK1/2 pathway blockers increased hypoxia-induced cell death [34]. ERK1/2 may also play a role in neuronal protection in status epilepticus. Inhibition of ERK1/2 activation with SL327 increased mortality in rats with pilocarpine-induced seizures [35]. Interestingly, in several non-neuronal systems, damaging stimuli including DNA injury, oxidative stress or death receptor signaling were reported to mobilize an anti- apoptotic ERK1/2 activity [36–38]. These data suggest an interesting possibility that ERK1/2 activation may be a general defense mechanism that is mobilized to protect different cell types against various forms of damage. Protective mechanisms downstream of ERK1/2 The ERK1/2 pathway affects multiple targets that may mediate its prosurvival activity. These include transcription factors that could stimulate production of anti-apoptotic mediators or inhibit accumulation of killer proteins. In addition, ERK1/2 may also directly affect several cell death/ cell survival regulators. The protective mechanisms that do not involve gene expression may be particularly relevant for the survival of damaged neurons, under conditions where gene expression machinery is disturbed. In the nervous system, protective ERK1/2 signaling may target both gene expression-dependent and gene expression- independent events (Fig. 1). For instance, Bonni et al. suggested that activation of a transcription factor, cAMP response element binding protein (CREB) and/or a direct inhibition of Bad, a pro-apoptotic member of the bcl-2 family, may mediate the prosurvival activity of ERK1/2 in trophic-deprived cerebellar granule neurons [12]. In this study, ERK1/2 appeared to mediate its effects through direct action on its target kinase, p90Rsk2. Inhibition of Rsk2 with a dominant-negative mutant blocked ERK1/2- mediated inhibition of apoptosis. Also, the prosurvival activity of ERK1/2 was reduced by antagonizing the Rsk target CREB. In addition, a dominant-negative mutant form of Rsk2 decreased the anti-apoptotic phosphorylation of Bad at its Ser112 residue. Finally, Rsk2 activation by ERK1/2 protected against apoptosis that was induced by an overexpressed wild type, but not a Ser112 fi Ala mutant form of Bad. Inhibitory Bad phosphorylation is also implicated as a mechanism of prosurvival ERK1/2 activity in neurons exposed to damage. In cultured hippocampal neurons that were protected against staurosporine-induced apoptosis by TGFb1, ERK1/2 stimulated survival by Fig. 1. Neuroprotective mechanisms employed by ERK1/2. Bold italic indicates the targets whose regulation by ERK1/2 appears to be gene expression-dependent. ERK1/2-mediated inhibition of Bim may involve both inhibitory phosphorylation and decreased expression. Dotted lines indicate prosurvival ERK1/2 targets that were found in non-neuronal systems and may also be involved in neuroprotection. See text for more details. 2052 M. Hetman and A. Gozdz (Eur. J. Biochem. 271) Ó FEBS 2004 Rsk-mediated phosphorylation of Bad Ser112 [29]. More- over, in murine brain, TGFb1 protected against ischemia while activating ERK1/2 and increasing Bad phosphoryla- tion at Ser112 [29]. This same ERK/Rsk-mediated phos- phorylation event also occurred in cultured mouse cortical neurons that were exposed to a neuroprotective hypoxia treatment [34]. In neuronally differentiated PC12 cells, the anti-apoptotic protection with NGF was suggested to be a result of ERK1/2-mediated inhibition of another pro-apoptotic member of the bcl-2 family, Bim40. The increase in Bim expression was implicated as one of the killer mechanisms activated by NGF withdrawal in this system. If trophic- deprived cells were rescued with NGF, Bim protein levels decreased. This effect was mediated by the ERK1/2 pathway [39]. Furthermore, ERK1/2 activation induced Bim phosphorylation at Ser109 and Thr110 residues that inhibited apoptosis induced by the overexpressed Bim40. Therefore, it appears that the ERK1/2 pathway may inhibit Bim function both by the decrease in Bim expression and by the direct phosphorylation of Bim. Neuroprotection by ERK1/2 may also involve a CREB- mediated increase in the expression of anti-apoptotic members of the bcl-2 family including bcl-2 and bag-1. For instance, the prosurvival activity of ERK1/2 in PC12 cells may proceed via CREB-stimulated expression of bcl-2 [40]. Furthermore, in HSV2-infected neurons, apoptosis was suppressed by an ERK1/2-dependent increase of bag-1 expression [30] that was presumed to be CREB-mediated. Similarly, overexpression of the endogenous CREB antag- onist ICER induced apoptosis and decreased bcl-2 levels in cultured rat cortical neurons [41]. Finally, ERK1/2 may protect from neuronal death by inhibiting the activity of a pro-apoptotic kinase GSK3b [42]. Neuroprotective mechanisms controlled by ERK1/2 may also involve indirect effects in non-neuronal CNS cells including glia and vascular cells. For instance, Gabriel et al. have shown that in mixed cortical cultures, neurons were protected from NMDA-mediated excitotoxicity by TGFa- induced ERK1/2 activation in astrocytes [43]. In these cells, ERK1/2 increased production of type 1 inhibitor of tissue type plasminogen activator that blocked the neurotoxic activity of a secreted protease, tissue-type plasminogen activator. Interestingly, Notch3 protects against ischemic stroke by supporting survival of brain vascular smooth muscle cells [44]. Notch3 mediated protection is suggested to occur through an ERK1/2-dependent up-regulation of c-FLIP that subsequently blocks the pro-apoptotic activa- tion of caspase 8 [44]. Outside the nervous system, there are several interesting observations pointing to the possible modes of ERK1/2- mediated neuroprotection. Allan et al. found that ERK2 can directly phosphorylate caspase 9 at Thr125 [45]. This phosphorylation event blocked caspase 9-mediated activa- tion of caspase 3 [45]. Indeed, there are reports suggesting that ERK1/2 may protect by inhibiting apoptosis down- stream of cytochrome c release, one of the key steps directly linked to caspase activation in the mitochondrial death pathway [46,47]. Another example of an interesting anti-apoptotic ERK1/2 target is a protein encoded by the Drosophila head involution defective (Hid) gene. Hid activates Drosophila caspases and mammalian caspase 8 [48,49]. Activity of this protein is inhibited through a direct phosphorylation by ERK1/2 [48]. Intriguingly, caspase 8 activation by death receptor signaling was blocked through ERK1/2 [38]. This inhibition was independent of protein synthesis indicating that a Hid-like ERK1/2 target may exist in mammals [38]. Noteworthy, in cardiomiocytes, ERK1/2 protected against cell death by the upregulation of cyclooxygenase-2 gene transcription and the resulting increase in production of cytoprotective prostaglandins [36]. A functional proteomics approach revealed several candidate ERK1/2 signaling substrates that may participate in neuroprotection [50]. For instance, ERK1/2 activation targeted an anti-apoptotic member of the bcl-2 family, mcl-1 and also DNA repair enzymes of the nucleotide excision repair pathway [50]. The significance of these ERK1/2 signaling substrates for neuronal survival remains to be elucidated. Lastly, ERK1/2 activation may be beneficial for functional recovery following CNS injury in the absence of eliciting any effects on the survival of damaged neurons. For instance, ERK1/2 may enhance proliferation of neural stem cells that may replace the deceased neurons [51]. ERK1/2 may also contribute to the functional plasticity after traumatic brain injury [52]. In conclusion, dissection of the protective mecha- nisms activated by ERK1/2 in the nervous system is still incomplete. ERK1/2: Killer or savior? There are several reports summarized in the accompanying review by Chu et al. that ERK1/2 inhibition protects neurons in ischemia, traumatic brain injury, epilepsy or oxidative glutamate toxicity [53]. How does one reconcile these observations with the proposed protective role of ERK1/2? Interestingly, several signaling systems have also been recognized to have seemingly contradictory effects on cellular survival. For instance, NMDAR can trigger cell death but may also have protective effects [54]. Likewise, the tumor suppressor protein p53 can trigger apoptosis but may also enhance DNA repair and cell survival [55]. The factors that are proposed to determine the beneficial or deleterious outcome of these multifunctional transducers include differences in the activation intensity or duration, the subcellular localization of signaling molecules, the signaling context provided by other pathways or the cellular energetic state. Importantly, the nature and extent of cellular injury may also change the ultimate results of the same signaling events. Finally, chronic activation of a signaling pathway may lead to an increased activity of inhibitory feedback pathways switching off downstream signaling that is normally activated by this circuitry. Some or all of these factors may also affect the ultimate outcome of ERK1/2 activation in the nervous system. A number of these issues are further discussed in the accompanying review by Chu et al. with a particular focus on neuro- degeneration [53]. Perspectives In summary, it appears that in many cases ERK1/2 activation is neuroprotective and mediates the effects of Ó FEBS 2004 Role of ERK1/2 in neuronal survival (Eur. J. Biochem. 271) 2053 several extrinsic survival signals. Furthermore, ERK1/2 activation is found in injured neurons where, at least in some cases, it has the protective, compensatory role. The neuroprotective mechanisms controlled by ERK1/2 include regulation of pro- and anti-apoptotic members of the bcl-2 family. It is probable that other mediators underlying prosurvival activity of ERK1/2 in neurons will be uncovered in the future. The factors that determine whether ERK1/2 activation will stimulate or inhibit neuronal survival will also be an interesting target for research. Identification of these factors may be critical for the development of useful strategies for targeting the ERK1/2 cascade to intervene against neurological diseases. Acknowledgements This work was supported by a NIH/NCRR grant, RR15576. 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EMBO J. 20, 914–923. Ó FEBS 2004 Role of ERK1/2 in neuronal survival (Eur. J. Biochem. 271) 2055 . MINIREVIEW Role of extracellular signal regulated kinases 1 and 2 in neuronal survival Michal Hetman 1, 2, 3 and Agata Gozdz 1 1 Kentucky Spinal Cord Research Injury Center and Departments of. transforming growth factor a. (Received 14 February 20 04, accepted 18 March 20 04) Eur. J. Biochem. 2 71, 20 50 20 55 (20 04) Ó FEBS 20 04 doi :10 .11 11/ j .14 32 -10 33 .20 04.0 413 3.x overexpression of mutated. neuroprotective interventions in diseases. ERK1 /2 signaling ERK1,alsoknownasp44mitogenactivatedproteinkinase (MAP kinase), and ERK2 (p 42 MAP kinase) are closely related protein kinases of the MAP kinase