Src family of kinases: protective or aggravating factor for ischemia reperfusion injury in nervous system?

Review

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Introduction

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Conclusion

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Expert opinion

FAK/Src family of kinases: protective or aggravating factor for ischemia reperfusion injury in nervous system? Christos Bikis, Demetrios Moris†, Ioanna Vasileiou, Eustratios Patsouris & Stamatios Theocharis

Expert Opin. Ther. Targets Downloaded from informahealthcare.com by Universitat de Girona on 12/12/14 For personal use only.

National and Kapodistrian University of Athens, Athens, Greece

Introduction: The focal adhesion kinase (FAK) and the Src families of kinases are subfamilies of the non-receptor protein tyrosine kinases. FAK activity is regulated by gene amplification, alternative splicing and phosporylation/ dephosphorylation. FAK/Src complex has been found to participate through various pathways in neuronal models of ischemia-reperfusion injury (IRI) with conflicting results. The aim of the present review is to summarize the currently available data on this subject. Areas covered: The MEDLINE/PubMed database was searched for publications with the medical subject heading IRI and FAK and/or Src, nervous system. We restricted our search till 2014. We identified 93 articles that were available in English as abstracts or/and full-text articles that were deemed appropriate for our review. Expert opinion: FAK has been found to have a beneficial preconditioning effect on IRI through activation via the protein kinase C (PKC) pathway by anesthetic agents. Of great importance are the interactions between FAK/Src and VEGF that has been already detected as a protective mean for IRI. The effect of VEGF administration might depend on dose as well as on time of administration. A Ca2+/calmodulin-dependent protein kinase II or PKC inhibitors seem to have protective effects on IRI by inhibiting ion channels activation. Keywords: focal adhesion kinase, ischemia-reperfusion injury, nervous system, Src Expert Opin. Ther. Targets [Early Online]

1.

Introduction

The Focal adhesion kinase (FAK) family of kinases is a subfamily of the nonreceptor protein tyrosine kinases (PTKs), counting two members, FAK and FAK2. Src family of kinases (SFK) is another non-receptor PTK subfamily, which is characterized by morphological similarity to FAKs [1]. Ischemia reperfusion injury (IRI) refers to the damage caused to tissues that have been deprived of blood supply, after blood circulation is restored. While injury as a result of ischemia (ischemic injury) seems a relatively easy to deduce consequence, the notion of damage caused by restoring nutrient and oxygen supply -- Reperfusion Injury -- might at first seem paradoxical. Nevertheless, IRI constitutes a major clinical problem that occurs as an outcome of diverse situations and can practically affect any tissue or organ. More specifically, its effects can vary from subclinical, cellular-level damage to acute vital organ deficiency and death. Almost any human cell is susceptible to IRI, which makes it easy to propose some common, basic mechanisms of this phenomenon. On the other hand, it still remains difficult to elucidate those mechanisms to their full extent for every different cell, tissue and organ, due to the numerous interactions that occur when moving from simple in vitro to complex in vivo models. 10.1517/14728222.2014.990374 © 2014 Informa UK, Ltd. ISSN 1472-8222, e-ISSN 1744-7631 All rights reserved: reproduction in whole or in part not permitted

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Ischemia-reperfusion injury (IRI) constitutes a major clinical problem affecting any tissue or organ but it still remains difficult to elucidate those mechanisms involved to its development to their full extent for every different cell, tissue and organ. Focal adhesion kinase (FAK)/Src complex activity has a crucial role in cell--extracellular matrix interaction and the transduction of extracellular signals and thus regulating cellular behaviors such as migration, proliferation and differentiation. FAK is essential to integrin signaling and once activated by integrin or non-integrin stimuli, such as cell adhesion and stimulation by growth factors, binds to a series of molecules activating them and initiating a series of signaling pathways. The most prominent integrin, whose action is associated with FAK, is b1-integrin. The FAK family of kinases in the pathogenesis of IRI is probably biphasic and complex, since they may take part in a series of a series of pathways that may at first seem to have opposing actions. A number of anesthetic agents such as anandamide, thiopental, isoflurane and sevoflurane activate FAK via the PKC pathway and thus have a beneficial preconditioning effect on IRI.

This box summarizes key points contained in the article.

The aim of the present review is to elucidate the role of FAK/SFKs during IRI in the nervous system. 2.

Body

Materials and methods We performed an electronic search through PubMed/Medline database by using the key terms: FAK*, Src family*, SFK*, organ*, IRI* and system*. The initial relevant studies retrieved from the literature were 249 from PubMed. This number was initially limited, after excluding the reviews and studies reporting data for pathways other than FAK, to number articles. After going through the manuscripts found from this search, we excluded studies that did not fulfill the criteria -- as far as all the necessary variables or values is concerned -- for analysis to our review. The final number of articles included in the review about ‘role of FAK in IRI’ were 93. Two independent reviewers (CB and DM) performed the literature search, the study selection and the data extraction. There was no time or publication limit in our literature search, whereas all the references from the identified articles were also searched for relevant information. 2.1

2.2

General principles The FAK family of kinases

2.2.1

FAK is a key regulator of growth factor receptor- and integrinmediated signals, governing fundamental processes in normal and diseased cells through its kinase activity and scaffolding function [1,2]. Although it was originally characterized as a 2

constituent of focal adhesions in fibroblasts, FAK is now considered to be not only a mediator of adhesion processes but also a crucial regulator of guidance and a modulator of gene expression [3]. FAK is the main transducer of the integrin signaling required to stabilize the actin cytoskeleton. Moreover, its signaling involves not only phosphorylation but also ubiquitination and proteolysis [3]. It is the first member of the family of FAK kinases to be discovered, first described in 1992 [4], and it is located at the focal adhesion sites (FAS) that play a predominant role in stabilizing endothelial cells at the extracellular matrix (ECM) [5]. Although the FAK cDNA codes for a protein of 119 -- 121 kDa depending on different species, it is known as p125FAK, as if it had a molecular weight of 125 kDa, a name which is based on its gel migration signature. FAK, which is an evolutionary conserved protein, has been mapped on the human chromosome 8 and is expressed in a wide variety of cells, with the degree of its expression ranging considerably [5]. The structure of FAK is unique among PTKs in comprising of a central kinase domain flanked by two large non-catalytic sections, the NH2-terminal region and the COOH-terminal region, while at the same time having no SH2 or SH3 binding domains, unlike other PTKs [5]. It does have SH2 and SH3-domain interacting phosphotyrosines and proline-rich regions, respectively, allowing it to activate with the downstream binding molecules [5]. FAK is essential to integrin signaling and once activated by integrin or non-integrin stimuli, such as cell adhesion and stimulation by growth factors, it binds to a series of molecules activating them and initiating a series of signaling pathways. The most prominent integrin, whose action is associated with FAK, is b1-integrin, but FAK can also be activated by b3 and b5 integrins [6]. Integrin activation and clustering leads to the binding of FAK to the cytoplasmic domain of activated b1-integrins and its subsequent autophosphorylation on tyrosine residue Y397, creating a special binding site for the SH2 domain of the SFKs [6-8]. After binding to FAK, Src phosphorylates the first at several domains, including tyrosine residues Y576/Y577 and Y861 further increasing its activity as a kinase [7]. The formation of this bipartite complex results in the enzymatic activation of both kinases, which in turn can act on and interact with a number of molecules [7,9]. The downstream molecules activated by the FAK-Src complex through phosphorylation include p130Cas (a docking protein), growth factor receptor-bound protein-2, phosphoinositide-3 kinase (PI3K) and paxillin (a cytoskeletal/adapter protein) [9,10]. The other member of the FAK family of kinases, named FAK2, or PYK2 (proline-rich tyrosine kinase 2), or cell adhesion kinase, is a 116 kDa cytoplasmic kinase highly expressed in the CNS, which has been shown to follow the same basic principles with FAK. PYK2 activation results from its autophosphorylation on Tyr-402, thus creating an SFK binding site and creating the FAK2/Src complex, whose mechanism of action is similar to the aforementioned FAK/Src complex.

Expert Opin. Ther. Targets (2014) 19(4)


FAK/Src family of kinases: protective or aggravating factor for ischemia reperfusion injury in nervous system?

Under normal circumstances, FAK activity is carefully regulated by gene amplification, alternative splicing and phosporylation/dephosphorylation. Moreover, due to its location and its affinity with integrins, FAK/Src complex activity plays a predominant role in cell-ECM interaction as well as the transduction of extracellular signals across the plasma membrane, regulating cellular behaviors such as migration, proliferation and differentiation [6,10]. The importance of this delicate balance of mechanisms that exists in normal cells is evident in the case of oncogenesis [4]. Tumor cells are characterized by disrupted, inhibited or unchecked FAK signaling which promotes malignant characteristics, such as overproliferating, antiapoptotic, invasive adhesive and migrating qualities, coupled with tumor angiogenesis and metastasis abilities [2,4,10,11]. The contradictory remarks that both FAK induction or inhibition can result in malignant phenotypes further prove that FAK/Src activation is the first step in a series of signaling pathways that regulate a considerable number of widely diverse cellular functions. Another field where the data on FAK action are controverisal is the aspect of cytotoxicity. While cytotoxic substances such as arsenic (As), lead (Pb), acrylamide, methylisothiazolinone, dichlorovinylcysteine and halothane have been proven to exert their action at least partially through downregulation of FAK phosphorylation [12,13], the bacterial toxins Pasteurella multocida toxin and Escherichia coli cytotoxic necrotizing factor have been shown to induce cytotoxicity by the exact opposite mechanism, namely the increased FAK phosphorylation. an and b1 integrins mediate Ab-induced neurotoxicity in primary hippocampal neurons by causing Ab-induced apoptosis [14]. The underlying pathogenesis may be related to activation of tyrosine-phosphorylation by FAK [14]. Inhibition of the interaction between Ab and Itgs or the interruption of FAK activation may effectively inhibit apoptosis in hippocampal neurons in diseases such as Alzheimer’s disease (AD) pathogenesis [14]. Ischemia reperfusion injury IRI refers to the damage caused to tissues that have been deprived of blood supply, after blood circulation is restored. As we have already mentioned, IRI can be caused by a variety of conditions with a variety of presentations and affect any tissue or organ. Adding to the complexity of the matter at hand, IRI is considered to be able to activate both apoptotic and anti-apoptotic or proliferating, invading and migrating pathways, alternatively or at once, with the end result depending on a number of factors that remain to be clarified. A series of hypotheses have been given concerning the pathogenesis of IRI, ranging from the dysfunction of the mitochondrial system and the mobilization of calcium and the depletion of ATP at cellular level, to the disruption of cell adhesion and cytoskeletal network at tissue level. At cellular level, oxidative stress plays the pivotal role in IRI. The creation of reactive oxygen species (ROS) after the reperfusion and re-oxygenation of a formerly ischemic tissue has been already proven in brain cell models [11,13,15,16]. Three 2.2.2

main sources of ROS during IRI exist: i) the mitochondrial respiratory complex, mainly in non-phagocytic cells [16,17]; ii) the (Nicotinamide adenine dinucleotide phosphate oxidase)-Nox family of oxidases [15,18]; and iii) other sources, such as the neutrophil-specific myeloperoxidase and thepresent in the liver-xanthine oxidase enzymes [17-20]. After the onset of ischemia, as well as following reperfusion, a series of signaling pathways are set in motion, with the family of MAPK having already been recognized as mediators of significant importance [20,21]. Apart from the production of ROS at cellular level, the immune system seems to be a contributing factor to IRI at tissue level, as far as cell adhesion is concerned. It seems logical to assume that the compromised cytoskeletal network integrity and the disrupted cell adhesion observed in IRI can only be explained by a multi-factor model, including a potentially large number of the proteins clustered at FAS. The ATP depletion observed in IRI, serves as a further argument to this case, because ATP-dependent protein phosphorylation is one of the principal regulatory mechanisms in the-rich with protein complexes-FAS. Role of FAK in IRI The seemingly bipolar action of the FAK/Src complex is also evident in the case of IRI (Figure 1). It has already been proven that the increased adhesion of polymorphonuclear neutrophils to endothelial cells, following the production of ROS coincides with the increased phosphorylation of several proteins located at the FAS, such as the cytoskeletal proteins paxillin and p130 cas, as well as several tyrosine kinases, which have been identified as members of the FAK/Src family [9,19]. On the other hand, while increased ROS production leads to increased FAK production, ischemia by itself causes FAK cleavage and depletion from FAS, leading to the conclusion that the involvement of the FAK family of kinases in the pathogenesis of IRI is probably biphasic and complex, since they may take part in a series of pathways that may at first seem to have opposing actions [21]. Furthermore, the reaction of the FAK/Src complex during IRI can vary significantly between different systems of the human organism and even in different parts and tissues of the same organ, rendering a more detailed and individualized approach necessary. 2.2.3

Nervous system and IRI The role of FAK/Src in neuronal models of IRI has been the object of considerable research, which in some cases leads to consistent results and in others, at least primarily, and contradicting observations. It should also be pointed out expressly that FAK and its similar PYK2 interact differently with each type of neuronal cells [21,22]. It is easier thus to observe these interactions separately as far as that is possible. Normally, the FAK/Src complex has been found to work through a series of pathways, one of which most probably includes the association with p130Cas, with the activation of extracellular-signal-regulated kinases (ERK) through the 2.3


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ROS

Intracellular Ca2/PKC

ECM/integrins

VEGF

FAK


Src

Cell differentiation

Cytoskeletal proteins

Nedd9 gene

p130 cas, paxillin Cell survival PI3K/Akt MARK ERK/Fyn tyrosine kinase Akt/NF-kB

Figure 1. An illustration of the role of the FAK/Src complex in the case of ischemia reperfusion injury. ECM: Extracellular matrix; FAK: Focal adhesion kinase; Nedd9: Neural precursor cell expressed, developmentally down-regulated 9; PI3K: Phosphoinositide3 kinase; PKC: Protein kinase C; ROS: Reactive oxygen species.

Fyn tyrosine kinase, an effect that finally promotes cell viability [22-24]. Promotion of cell survival was also found to be triggered by the cells’ attachment to ECM components, leading to engagement of the integrins and FAK phosphorylation, which would then regulate the interaction of cytoskeleton actin-bound with the neuronal membrane and also function through the PI3K kinase/Akt and Akt/NF-kB pathway promoting cell survival [24-29]. FAK activity, in vitro, was also found to be regulated by signals initiated by growth factor/cytokine receptors and a number of G-protein-coupled receptors [30-32]. Table 1 summarizes the role of FAK in IRI. The importance of FAK/Src system for cell signaling is prominent through the modifications induced by diverse pharmaceutical substances. Various extracellular signals that increase the intracellular Ca2+ lead to the protein kinase C (PKC) family activation and thus, the stimulation of FAK via phosphorylation [25]. However, while a number of anesthetic agents such as anandamide, thiopental, isoflurane and 4

sevoflurane activate FAK via the PKC pathway and thus have a beneficial preconditioning effect on IRI [33-35], another FAK phosphorylation pathway exists, with activation of the a2-adrenoceptor adenylate cyclase pathway, as it was observed in the case of canabinoid 1 (CB1) receptors which are negatively coupled to adenylate cyclate [36-38] and the beneficial preconditioning effect of dexmedetomidine, which activates the a2-adrenoceptor-adenylate cylcase pathway, in IRI models [39]. Recently, Dalton et al. [38] showed that CB1 receptor agonists failed to stimulate FAK Tyr-P in the absence of integrin activation in neuronal cells. In contrast, in integrin presence, ligands such as fibronectin and laminin displayed increased FAK 576/577 Tyr-P that was augmented by CB1 receptor agonists and blocked by the Src inhibitor PP2 and Flk-1 VEGFR antagonist SU5416 (Table 2) [38]. On the most classic IRI model, that of ischemia caused by short-time selective vessel occlusion, followed by reperfusion, some of the observed results are the decrease of FAK Tyr-397 phosphorylation (after 24 -- 72 h, probably initiated by ATP depletion), the decrease of the FAK/Src interaction complex, the decrease of the total amount of FAK, the decrease of laminin levels and the increased MMP activity, while there is no effect on the FAK/p130Cas complex and the MMP expression [40,41]. The degradation of ECM proteins such as laminin alters and disrupts the cell adhesion to the ECM, thus leading to a decrease of the FAK phosphorylation and a subsequent decreased FAK/Src association, a prelude to cellular apoptosis [40,42]. Furthermore, the decrease of the total amount of FAK could be up to a part caused by the overactivation of calpain and caspaces, a finding on non-neuronal cells that is possible to additionally apply to neuronal ones [43-47]. One other equivalent IRI model is that of chemical insult, which has been found to lead to generation of ROS, activation of phosphotyrosine phosphatases and subsequent inhibition of SFKs by disassembly, inhibition and degradation of FAK, as well as disassembly of the FAK/Src complex [48]. On the other hand, some upstream or downstream effects of IRI and FAK/Src deserve more attention than others such is the case with VEGF. In normal conditions, interaction between VEGF and integrin avb3 leads to FAK phosphorylation, which according to what has been already mentioned would mean that upregulation of VEGF serves as a protective mean for IRI [49-51]. On the other hand, things are more complicated than they seem. Studies that reveal favorable effects of blocking VEGF before IRI [52,53], show that the mechanism is more complex and other parameters should also be taken into account. During IRI, treatment with the VEGF inhibitor cyclo[Arg-Gly-Asp-D-Phe-Vall] (cRGDFV), resulted in a decrease in the inflammatory cells recruitment and also to a decreased FAK phosphorylation, possibly due to the blocking of the interaction between the integrin avb3 (upregulated selectively after brain ischemic injury) and the peptide sequence argine-glycine-aspartic acid (RGD) [54,55], an event that could obstruct FAK/integrin association and thus the phosphorylation of the former. As an end result of cRGDFV




Table 1. Summary of the role of FAK in ischemia-reperfusion injury. Author

Year

Study design

Mechanism of action

Result

Model

Schlaepfer et al. [22]

1998

Experimental study

Promotes cell viability

Igishi et al. [24]

1999

Experimental study

FAK/Src complex work through p130Cas and with the activation of ERK through the Fyn tyrosine kinase Activated FAK stimulated ERK and JNK activity by inducing tyrosine phosphorylation of Shc

Lin et al. [23]

2004

Experimental study

FAK binds to the b1 integrin cytoplasmic domain, and subsequently binds to the SH2 domain of c-Src, as well as p130cas

Cell spreading, motility and proliferation

King et al. [29]

1997

Experimental study

The association of (PI) 3-kinase with FAK was induced by the attachment of fibroblasts to fibronectin and was dependent on the SH2 domain of p85

Cell spreading, motility and proliferation

Tamura et al. [28]

1999

Experimental study

Cells’ attachment to extracellular matrix components, leads to engagement of the integrins and FAK phosphorylation

Promotion of cell survival

Integrin stimulation by fibronectin leads to changes in intracellular protein tyrosine phosphorylation events Fibronectin was used as a trigger to FAK activation. FAK to stimulate signaling events leading to the activation of ERK and JNK Following integrin occupancy, FAK is autophosphorylated at Y397, thereby creating a binding site for the SH2 domain of c-Src family kinases. The c-Src/FAK complex stabilizes c-Src catalytic activity. Activated c-Src then phosphorylates additional sites on FAK, including the regulatory loop tyrosines, Y576 and Y577, in the catalytic domain, thereby enhancing FAK activity Cell attachment to fibronectin stimulates the integrindependent interaction of p85-associated PI 3-kinase with integrin-dependent FAK as well as activation of the Ras/ mitogen-activated protein kinase pathway The interaction of cytoskeleton with the neuronal membrane and also function through the phosphoinositide-3 kinase/Akt and Akt/NF-kB pathway promotes cell survival

Cell stimulation

ERK: Extracellular-signal-regulated kinases; FAK: Focal adhesion kinase; PI: Phosphatidylinositol.

administration, focal cerebral ischemic damage was ameliorated [56]. It was also shown that the mechanism of VEGF from recruited inflammatory cells, promoted survival instead of apoptosis, as would have been expected from the FAK dephosphorylation result alone. To that avail, the protective effects of soluble flt-1 gene transfer, a natural VEGF inhibitor are of interest [57]. Last but not least, one should also take into account the fact that during ischemia, VEGF interacts directly with Src, increasing vascular permeability and thus aggravating the ischemic damage [58]. Therefore, blockade of Src reduces neuronal damage after brain ischemia [59]. To sum up, it seems that during ischemia, VEGF interaction leads to acute angiogenesis, FAK phosphorylation and also activation of other, contradicting damage inducing mechanisms. As a result, VEGF administration after ischemia can either reduce or cause more damage depending on both concentration and time of administration. Up to now, it seems that in

the early phase of IRI is better to block VEGF rather than upregulate it. Another interesting observation, that correlates the FAK/ Src pathway not only with cell survival but also with neuronal differentiation is the observed effect of the neural precursor cell expressed, developmentally down-regulated 9 (Nedd9) gene on FAK/Src. Nedd9, a splicing variant of Cas-L (Crkassociated substrate lymphocyte-type) plays a role in neuronal differentiation that is normally not existent in the adult brain [8,60,61]. The Cas-L protein interacts and gets phoshporylated by both FAK and PYK2 [62], with IRI causing increased expression of both Nedd9 mRNA and FAK, as well as increased interaction between them [8]. As a result, after ischemia, Nedd9/FAK delayed expression was found to contribute to neuronal damage repair [8,63]. The effect of Nedd99 reveals the possible role that FAK/Src plays in neuronal differentiation. Although of the postnatal age has no effect on the


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Table 2. Summary of the studies referring to therapeutic interventions aimed to target FAK. Author

Year

Study design

Intervention

Mechanism of action

Results

Derkinderen et al. [35]

1996

Experimental study

Anandamide

The activation FAK via the PKC pathway has a beneficial preconditioning effect on IRI

Dahmani et al. [34]

2004

Experimental study

Thiopental and isoflurane

Anandamide is an endogenous ligand for cannabinoid receptors. It increases protein tyrosine phosphorylation in neurons. One of the proteins phosphorylated is FAK+ expressed preferentially in neurons Oxygen-glucose deprivation decreases the ATPdependent phosphorylation process of Focal Adhesion Kinase (pp125FAK)

Dahmani et al. [37]

2004

Experimental study

Thiopental, propofol, etomidate, isoflurane, sevoflurane and desflurane (anesthetic agents)

Dahmani et al. [39]

2005

Experimental study

Dexmedetomidine on phospho-tyrosine FAK phosphorylation

Burnett et al. [54] Nisato et al. [55]

2005

Experimental study

cyclo[Arg-Gly-Asp-D-Phe-Vall] (cRGDFV-VEGF inhibitor)

Liu Y et al. [69] Liu Y et al. [70,71]

2001

Experimental study

Ketamine (NMDA receptor selective antagonist) and Nifedipine (the L-voltage gated Ca2+ channel antagonist)

2003

2003

The anesthetic-induced increase in ppFAK phosphorylation was blocked by three structurally distinct inhibitors of PKC. gaminobutyric acid type A receptor antagonist and inhibitor of the ryanodine receptor were ineffective in blocking anesthetic-induced activation of tyrosine phosphorylation Dexmedetomidine is a potent and selective a2-adrenoceptor agonist that exhibits a broad pattern of actions, including sedation, analgesia and neuroprotection In normal conditions, interaction between VEGF and integrin avb3 leads to FAK phosphorylation, which means that upregulation of VEGF acts protectively in IRI Glutamate as well as cell membrane depolarization have been shown to increase Src activation and thus further increase the PYK2/Src interaction, which is also facilitated by the translocation of PKC, Src, Fyn and Pyk2 to postsynaptic density protein postischemia

Phosphorylated pp125FAK content was markedly decreased in neuronal tissue subjected to oxygen-glucose deprivation. Thiopental and isoflurane significantly attenuated this phenomenon, possibly via PKC activation Anesthetic agents markedly increase tyrosine phosphorylation of ppFAK in most likely via the phospholipase C-PKC pathway

Increase in phosphorylation of FAK via stimulation of a2 adrenoceptors and decrease in cleaved caspase-3 expression correlate with dexmedetomidine-induced cell survival Decrease in the inflammatory cells recruitment leads to a decreased FAK phosphorylation, possibly due to the blocking of the interaction between the integrin avb3 The end result is the activation of PYK2 by two independent pathways due to calcium influx, a PKC dependent one and a Ca2+/ CaM and CaMKII dependent one, independent to each other. KN62 (a CaMKII inhibitor) or CHE (a selective PKC inhibitor) will have a greater protective effect than Nifedipine or Ketamine alone

CaM: Calmodulin; CaMKII: Ca2+/calmodulin-dependent protein kinase II; FAK: Focal adhesion kinase; IRI: Ischemia-reperfusion injury; PKC: Protein kinase C; PYK: Proline-rich tyrosine kinase.

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amount of FAK and p130Cas, Src was found to increase with maturation by Zalewska et al. [64]. In the same frame, the same research team [40] revealed a very interesting temporal biphasic pattern with regard to FAK/Src activity after IRI. After its onset, IRI leads to activation of the energydependent ion channels resulting in loss of the membrane potential. Cell depolarization is then the trigger for the activation of voltage gated Ca2+ channels (VGCC), as well as the cause of neurotransmitters’ release (e.g., excitatory aminoacids) which then accumulate in the synapse and lead to activation of ligand-gated calcium channels, with the characteristic example of NMDA [65-67]. The openness of both types of channels, leads to the aforementioned influx of Ca2+, which has a dual effect. First, it leads to the activation of PYK2 in a PKC-dependent manner and the subsequent PYK2/Src complex formation. Second, it acts as the onset of further calcium-dependent signaling cascades which cause an early efflux of glutamate, which further activates the NMDA receptors, creating a positive feedback circuit [68]. Furthermore, glutamate as well as cell membrane depolarization have been shown to increase Src activation and thus further increase the PYK2/Src interaction, which is also facilitated by the translocation of PKC, Src, Fyn and Pyk2 to postsynaptic density protein (PSD) postischemia [69-75]. That can also been proven by observing the effect of Ketamine (NMDA receptor selective antagonist) and Nifedipine (L-VGCC antagonist) have on PYK2 activation, also suggesting a possible cross-talk between the NMDA and the L-VGCC channels by returning back through the proposed closed loop [69,76,77]. The end result is the activation of PYK2 by two independent pathways due to calcium influx, a PKC-dependent one and a Ca2+/Calmodulin (CaM) and CaM-dependent protein kinase II (CaMKII) dependent one, independent to each other [77]. It is also noteworthy to point out that the later on we target the described pathway with an inhibitor, the greater will the percentage of final loss of PYK2 activity will be, that is, KN62 (a CaMKII inhibitor) or CHE (a selective PKC inhibitor) will have a greater protective effect than Nifedipine or Ketamine alone. Interestingly, CHE also has another blocking capability, namely preventing the Src-induced phosphorylation of NMDA receptor subunit 2A (NR2A), NR2B which would cause our positive feedback circuit that was mentioned above, also indicating PKC involvement in the NR2A, NR2B phosphorylation [78]. Last but not least, it should be noted that although this mechanism generally holds true at a structural level, the different times of activation for individual pathways could vary in different cells’ tissue types, therefore explaining the following finding of Liu et al. [71] who referred that 1 h after the onset of focal cerebral ischemia, PYK2 activity was observed mainly in the cortical neurons, whereas after 24 -- 72 h PYK2 was mostly evident in microglia around the infracted area, suggesting a second-step microglia activation as part of the recovery/death mechanism.

The consequences of the aforementioned cascade web in detail are as follows: First of all, the activated PYK2 and PSD-95 lead to Src activation and the onset of the Src-MAPK signaling pathway which promotes cell apoptosis [69,78-84]. These apoptotic-leading MAPK pathways, which require G-protein-coupled receptors, include the p38 MAPK pathway, the c-Jun-N-terminal kinase pathway and the ERK pathway [85]. Especially, as far as the ERK is concerned, there seems to be a need for more research, since Corvol et al. [86] noted that PYK2 and ERK were activated at different cellular compartments after KCl depolarization of Hippocampal slices, although that finding could also be attributed to the methodological differences of using KCl depolarization instead of the classical IRI model and hippocampal tissue slices instead of cell lines. In any case, it is important to consider the differences that arise between different cell types in a certain tissue and homogenous cell series. It was also demonstrated that autophosphorylation of PYK2 was dramatically decreased in fun -/- mice but unaltered by SFK inhibitors, suggesting that in the proposed model, Fyn plays a role in the control of PYK2, not limited to its, already known, catalytic activity. Apart from the Src/MAPK pathway activation, the activated PYK2/Src complex also performed NMDAR additional phosphorylation, both at NR2A and NR2B [84,87], mediated through the interaction with the PSD-95 protein [84,88]. PSD-95-mediated tyrosine phosphorylation of L-VGCC a1C subunits [89] as well as NMDAR additional phosphorylation lead to delayed neuronal death due to a vicious circle of Ca2+ influx. Finally, PSD-95-mediated downstream calcium-dependent nitric oxide production [77] may lead to ischemic and excitoxic neurodegeneration. At this point, apart from the various targeted inhibitors that are quite obvious by considering the described pathways, a special reference should be made with regard to lithium, which yields a multiple protective effect against the PYK2/ Src complex activation. First, it suppressed the PYK2/PSD95 interaction necessary for NMDAR additional phosphorylation. Second, it suppressed the NR2A/PYK2 interaction and the NR2A, NR2B phosphorylation also involved in NMDAR additional phosphorylation. And finally, it caused a decrease in the phosphorylation of both PYK2 and Src, thus limiting the total complex activity in general. 3.

Conclusion

Current data on FAK/Src and IRI remain limited, but clearly indicate the contradicting actions of FAK/Src in neuronal cells. The FAK family of kinases in the pathogenesis of IRI is probably biphasic and complex, since they may take part in a series of a series of pathways that may at first seem to have opposing actions. Anesthetic agents activate FAK via the PKC pathway and thus have a beneficial preconditioning


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effect on IRI. Nevertheless, further investigation is required in order to establish their possible contribution in human neuronal IRI.


4.

Expert opinion

It was clearly shown that FAK exerted a beneficial preconditioning effect on IRI through activation via the PKC pathway (e.g., anesthetic agents). Regarding the most common model of neuronal IRI, that induced by vessel occlusion, decreased FAK phosphorylation and a decrease in FAK/Src association leads to cellular apoptosis. Of great importance are the interactions between FAK/Src and VEGF that have been already detected as a protective mean for IRI. However, studies in neuronal IRI reveal favorable effects of blocking VEGF before IRI, via obstruction of FAK phosphorylation. It could be thus suggested that the administration of a VEGF-inhibitor could ameliorate the focal cerebral ischemia damage. Moreover, blockade of Src reduces neuronal damage after brain ischemia. Other studies, however, exist, which promote the protective role of VEGF in IRI. The results of VEGF administration might thus depend on concentration as well as on time of administration. PYK2/Src complex has also been found to induce apoptosis in non-neuronal cells after its activation. Regarding neuronal cells, a CaMKII or a PKC inhibitor seem to have protective effects on IRI by inhibiting ion channels activation. Furthermore, lithium seems to exert a protective effect on IRI by decreasing phosphorylation of PYK2 and Src. Moreover, as we have already presented [90], proteasomes modification could be an effective future therapeutic strategy for neuronal IRI due to its correlation with pathways such as NF-kB inactivation and the cytoprotective proteins endothelial nitric oxide synthase, whose role, in the IRI in neuronal cells, was described in this review article. Bibliography Papers of special note have been highlighted as either of interest () or of considerable interest () to readers. 1.

2.

3.

4.

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Acknowledgement C Bikis and D Moris contributed equally to this work.

Declaration of interest The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. dynamic exchange of the docking protein NEDD9 (neural precursor cell expressed developmentally down-regulated gene 9) at focal adhesions. J Biol Chem 2014;289(36):24792-800

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Christos Bikis MD, Demetrios Moris† MD, Ioanna Vasileiou MD PhD, Eustratios Patsouris MD PhD & Stamatios Theocharis MD PhD † Author for correspondence National and Kapodistrian University of Athens, Anastasiou Gennadiou 56, 11474, Athens, Greece Tel: +30 210 6440590; E-mail: [email protected]


Affiliation

11

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