MAPK Signal Transduction Underlying Brain Inflammation and Gliosis as Therapeutic Target.код для вставкиСкачать
THE ANATOMICAL RECORD 292:1902–1913 (2009) MAPK Signal Transduction Underlying Brain Inflammation and Gliosis as Therapeutic Target BOZENA KAMINSKA,* AGATA GOZDZ, MALGORZATA ZAWADZKA, ALEKSANDRA ELLERT-MIKLASZEWSKA, AND MACIEJ LIPKO Laboratory of Transcription Regulation, Nencki Institute of Experimental Biology, Warsaw, Poland ABSTRACT A majority, if not all, acute and progressive neurodegenerative diseases are accompanied by local microglia-mediated inﬂammation, astrogliosis, inﬁltration of immune cells, and activation of the adaptive immunity. These processes progress by the expression of cytokines, adhesion molecules, proteases, and other inﬂammation mediators. In response to brain injury or infection, intracellular signaling pathways are activated in microglia, which turn on inﬂammatory and antigen-presenting cell functions. Different extrinsic signals shape microglial activation toward neuroprotective or neurotoxic phenotype under pathological conditions. This review discusses recent advances regarding molecular mechanisms of inﬂammatory signal transduction in neurological disorders and in in vitro models of inﬂammation/gliosis. Mitogen-activated protein kinases (MAPKs) are a family of serine/threonine protein kinases responsible for most cellular responses to cytokines and external stress signals and crucial for regulation of the production of inﬂammation mediators. Increased activity of MAPKs in activated microglia and astrocytes, and their regulatory role in the synthesis of inﬂammatory cytokines mediators, make them potential targets for novel therapeutics. MAPK inhibitors emerge as attractive anti-inﬂammatory drugs, because they are capable of reducing both the synthesis of inﬂammation mediators at multiple levels and are effective in blocking inﬂammatory cytokine signaling. Small molecule inhibitors targeting of p38 MAPK and JNK pathways have been developed and offer a great potential as potent modulators of brain inﬂammation and gliosis in neurological disorders, where cytokine overproduction contributes to disease progression. Many of the pharmacological MAPK inhibitors can be administered orally and initial results show therapeutic beneﬁts in preclinical animal models. Anat Rec, 292:1902–1913, C 2009 Wiley-Liss, Inc. 2009. V Abbreviations used: AP-1 ¼ activator protein 1; APC ¼ antigen-presenting cells; ARE ¼ AU-rich element; ASK ¼ apoptosis signal-regulating kinase; ATF-2 ¼ activating transcription factor 2; CRE ¼ cyclic AMP-responsive element; CBP ¼ CREB binding protein; CsA ¼ ciclosporin A; ERK ¼ extracellular signal-regulated kinase; iNOS ¼ inducible nitric oxide synthase; ISRE ¼ interferon-stimulated response element; JNK ¼ c-Jun N-terminal kinase; LPS ¼ lipopolysaccharide; MCAO ¼ middle cerebral artery occlusion; MCP-1 ¼ monocyte chemoattractant protein 1; MAPKAP-K2 ¼ MAP kinaseactivated protein kinase 2/3; MEK ¼ MAP/ERK kinase; MKK ¼ MAP kinase kinase; MAP3K ¼ MAP kinase kinase kinase; MLK ¼ mixed lineage kinase; MMPs ¼ metalloproteinases; MyD88 ¼ myeloid differentiation factor 88; NIK ¼ NF-Binducing kinase; PAK ¼ p21-activated kinase; STAT ¼ signal transducers and activators of transcription; TAK1 ¼ transforming growth factor-activated kinase 1; TBP ¼ TATA box binding protein; TGF ¼ transforming growth factor; TLR ¼ Toll-like receptors; TNF ¼ tumor necrosis factor; TRAF ¼ TNF receptor-associated factor; TRIF ¼ Toll/IL-1R domaincontaining adaptor-inducing IFN-beta Grant sponsors: Ministry of Science and Higher Education (Poland); Grant number: COST B30N N401 0475 and PBZ/ MEiN/01/2006/32. *Correspondence to: Bozena Kaminska, Department of Cell Biology, Nencki Institute of Experimental Biology, 3 Pasteur Str., 02-093 Warsaw, Poland. Fax: 822-53-42. E-mail: email@example.com Received 18 February 2009; Accepted 28 July 2009 DOI 10.1002/ar.21047 Published online in Wiley InterScience (www.interscience.wiley. com). MAPK SIGNALING IN NEUROINFLAMMATION 1903 Key words: neurodegeneration; microglia activation; brain inﬂammation; gliosis; proinﬂammatory cytokines; Toll-interleukin-1 receptor superfamily; signal transduction; MAP kinases; transcription factors; AP-1; NF-jB; STAT; small molecule inhibitors INTRODUCTION Inﬂammation (from Latin inﬂamatio, to set on ﬁre) is a key process in the host defense system, the complex biological response of tissue to harmful stimuli, damaged cells, or irritants. The initial response to harmful stimuli within the central nervous system (CNS) is achieved by the activation of resident microglial cells and is followed by the production of inﬂammation mediators triggering the increased movement of blood-derived immune cells into the injured tissue (Hanisch and Kettenmann, 2007). A cascade of strictly controlled biochemical events propagates and matures the inﬂammatory response, involving various cells within the injured tissue, the local vascular system, and the immune system. Chronic inﬂammation leads to a progressive shift in the type of cells which are present at the site of inﬂammation and is characterized by both destruction and repair of the tissue. The loss of control and prolonged inﬂammation can lead to a number of neurological diseases, including neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, age-related macular degeneration, HIV-associated dementia, brain trauma, and stroke (Stoll et al., 1998; Danton and Dietrich, 2003; McGeer and McGeer, 2003, 2004; Van Eldik et al., 2007). Inﬂammation can be triggered by several mechanisms: 1) neurons release bioactive peptides in response to pain (Steinhoff et al., 2000); 2) damaged cells release intracellular proteins, such as heat-shock proteins (Basu and Srivastava, 2000) or the high-mobility group 1 protein (HMGB1) (Scafﬁdi et al., 2002), or purines such as ATP (Davalos et al., 2005) that trigger cytokine production; 3) shed or secreted products of microorganisms are sensed through binding to soluble (complement system) or cellsurface receptors such as Toll family members (Toll-like receptors, TLRs), peptidoglycan recognition proteins and scavenger receptors (Elward and Gasque, 2003; Martinon and Tschopp, 2005). Although inﬂammatory responses can differ in various diseases, there is a common spectrum of genes and endogenous mediators involved, including growth factors, inﬂammatory cytokines such as interleukin-1 b (IL-1b), tumor necrosis factor (TNF)-a, interleukin-6 (IL-6), chemokines [Fractalkine, macrophage inﬂammatory factor (MIP-1a,b, IL-8)], matrix metalloproteinases (MMPs), lipid mediators, and toxic molecules such as nitric oxide (NO) or free radicals (Cayrol et al., 2008). In addition to degradation of extracellular matrix, MMPs cleave cytokines (e.g., TNFa, FasL) and chemokines from tissue macrophages as well as from blood-derived monocytes, amplifying inﬂammatory reactions. Prostaglandin E2 and neutrophil-derived defensins recruit lymphocytes; leukotrienes attract antigen-presenting dendritic cells. Activated lymphocytes further activate macrophages to secrete proteases, eicosanoids, cytokines, reactive oxygen, and nitrogen intermediates (Nathan, 2002). The common pathophysiological hallmarks of neurodegenerative disorders are activation of microglia and astrogliosis, inﬁltration of immune cells, and activation of the adaptive immune system (Gehrmann et al., 1995; Stoll et al., 1998; Danton and Dietrich, 2003; McGeer and McGeer, 2003; Dudal et al., 2004; Block and Hong, 2005; Koistinaho and Koistinaho, 2005; Eikelenboom et al., 2006; Whitton, 2007). Massive trauma, postischemic or toxicity-related necrosis, hemorrhage can trigger inﬂammatory responses, and resident microglia respond rapidly to neuronal cell death (Morioka et al., 1993; Block and Hong, 2005; Hanisch and Kettenmann, 2007; Rogers et al., 2007). Murine and human microglia express all TLRs reported to date (with exception of TLR9 not detected in humans) and respond to typical TLR stimuli by expressing a panel of innate immune cytokines, and chemokines, as well as costimulatory and MHC class I/II molecules (Olson and Miller, 2004). Furthermore, damaged or overactive neurons release or leak purines, including ATP and UTP that activate corresponding receptors: mainly P2X4 and P2X7, but also P2Y2, P2Y6, and P2Y12 expressed on microglia (Ohsawa et al., 2007). Excessive neuronal release of glutamate not only leads to neuronal death but also activates microglia-expressing metabotropic glutamate receptors (Taylor et al., 2002, 2005). After initiation of inﬂammation, microglial cells not only secrete large amounts of neurotoxic or proinﬂammatory cytokines (e.g., IL-1, TNF-a, IL-12, INF-c, FasL) but also produce anti-inﬂammatory molecules such as IL-10 or IL-1 receptor antagonist (Feuerstein et al., 1997; Aloisi, 2001). Although the repertoire of synthesized cytokines, chemokines, and neurotrophic factors determines a ﬁnal outcome of inﬂammation, a massive production of cytokines such as IL-1b or TNF-a is detrimental. The inhibition of their secretion or activity with neutralizing antibodies or soluble cytokine receptors decreases neuronal damage (Barone et al., 1997; Schielke et al., 1998; Shohami et al., 1999; Rothwell and Luheshi, 2000; Boutin et al., 2001; Aggarwal, 2003; Rothwell, 2003; Simi et al., 2007). Cytokines produced by microglia can activate astrocytes which in turn become important contributors to inﬂammatory and immune responses within the brain (Benveniste, 1998; Farina et al., 2007). Astrocyte activation (reactive astrogliosis) accompanies neuronal damage and microglia activation after insults of different etiology, including stroke (Stoll et al., 1998). Reactive astrocytes proliferate, undergo hypertrophy of processes and upregulation of intermediate ﬁlaments (Pekny and Nilsson, 2005), and produce neurotrophic substances, as well 1904 KAMINSKA ET AL. Fig. 1. Activation of astrocytes and microglia in the brain following transient ischemia. Staining with isolectin B4 and immunohistochemistry for glial ﬁbrillary acidic protein (GFAP) reveals a strong microglial activation and astrogliosis in the ischemic cortex in response to tran- sient MCAO. Bar graphs show the number of stained cells in the ipsilateral cortex at indicated time after ischemia (means SEM, *P > 0.05, **P > 0.01). as proinﬂammatory (IL-1, IL-6) and cytotoxic cytokines (FasL, TNF-a) (Benveniste, 1998). Activated astrocytes can produce toxic molecules such as reactive oxygen species and NO (for a review see Stoll et al., 1998). Figure 1 illustrates an increase in the number and morphological alterations of lectin B4-positive microglia and GFAP-positive astrocytes in the ischemic hemisphere 72 hr after a middle cerebral artery occlusion (MCAO). The signiﬁcant increase in the number of lectin B4-positive microglia was observed as early as 6 hr after ischemia and progresses through the postischemic period (Fig. 1). TNFa and IL-1b are upregulated in ischemic brain lesions within 6–12 hr, primarily in microglial cells and at 24 hr in astrocytes (Zawadzka and Kaminska, 2005). Cytokines and chemokines released by microglia promote the recruitment of lymphocytes and macrophages. Inﬁltration of peripheral macrophages and lymphocytes is not restricted to infectious or autoimmune disorders of the nervous system (Greter et al., 2005), but occurs in response to cerebral ischemia and traumatic lesions. Lymphocytic inﬁltration was observed in MCAO and photochemically induced focal ischemia in the rat cortex (Schroeter et al., 1994; Jander et al., 1995). Studies of postischemic damage and neurological deﬁcits in mice deﬁcient in lymphocytes (Rag1-/-), CD4þ T cells, CD8þ T cells, B cells, or IFN-c revealed that inﬁltrating CD4þ and CD8þ T lymphocytes and production of IFN-c contribute to the inﬂammatory and thrombogenic responses, brain injury, and neurological deﬁcits (Yilmaz et al., 2006). Human studies indicate that stroke recurrence/ death rates were signiﬁcantly associated with increasing CD4þCD28-lymphocyte counts in peripheral blood (Nadareishvili et al., 2004), and accumulation of CD4þ T cells was detected in the brains of postmortem human PD specimens (Brochard et al., 2009). These results indicate an involvement of the adaptive immune system in progression of brain damage. CELL CULTURE MODELS OF INFLAMMATION AND GLIOSIS A detailed study of receptor-triggered signaling and effector functions of microglia in vivo is difﬁcult. Although, microglia comprise 5%–15% of the resident cell population of the CNS, current methods for isolating MAPK SIGNALING IN NEUROINFLAMMATION 1905 Fig. 2. MAPK signaling in proinﬂammatory activation of microglia in vitro. Microglial cells isolated as previously described (Zawadzka and Kaminska, 2005) were activated by an addition of 100 ng/mL LPS and 24 hr later labeled with the Coomasie blue (A) to visualize mor- phological transformation; note the amoeboid morphology of activated cells. LPS induces a rapid activation of MAPK in cultured microglial cells (B) and upregulates the expression of inﬂammation-related genes as evaluated by a quantitative real-time PCR (C). these cells from adult brain are laborious and give low yields. Thus, it is easier to prepare primary microglia derived from mixed glial cultures established from neonatal mice and rats. The studies by Carson’s group demonstrated that cultured microglia display a much more homogenous phenotype than in the brain, possibly due to the homogenous environment of the culture plate (Carson et al., 2007). However, the results from in vitro culture may not be fully predictive of in vivo biology because of the absence of environmental signals provided by healthy neurons and astrocytes. Despite of all limitations, recent protocols allow for the isolation and maintenance of microglia in cell cultures prepared from rodents. Typically, glial primary cultures are prepared from neonatal brains and involve propagation of microglia on astrocyte monolayer followed by shaking off microglial cells and plating them on culture dishes for nonadherent cells. A maintenance of microglial cells for additional 48 hr results in silencing and the appearance of a ramiﬁed morphology (Fig. 2). Various assays can be performed to conﬁrm macrophage-like functional activity, including morphological alterations, motility, phagocytosis, and inﬂammatory gene expression. BV-2, murine-immortalized microglial cells, frequently used in the studies of microglial function (Petrova et al., 1999), under basal conditions are partly activated and do not recapitulate all features represented by primary microglia cultures. Cultured microglial cells retain their plasticity as indicated by their ability to upregulate MHC class II, differentiate into cells with a macrophage morphology following the addition of IFN-c and GM-CSF, and synthesize proinﬂammatory cytokines and inﬂammation modulators (iNOS, Cox2) (Carson et al., 2007). When stimulated with lipopolysaccharide (LPS), a bacterial wall component and inducer of monocytes/macrophages (alone or in combination with IFNc) microglial cells develop a potent neurotoxic phenotype (Bal-Price and Brown, 2001). Cultured microglia cells can also be used to model Alzheimer-related degeneration. b-Amyloid (Abeta) has been shown to induce iNOS in cultured microglia and kill cocultured neurons via an NO-dependent mechanism (Wisniewski and Wegiel, 1994). Fibrillar Abeta1-42 peptides induced the expression of iNOS, cytokines (TNF-a, IL-1b, and IL-6), and integrin markers in mouse primary microglia via TLR2 receptors (Jana et al., 2008). The establishment of protocols for pure primary microglial cultures grossly facilitated studies of mechanisms of microglial activation and functions involved in a variety of CNS pathologies. Rodent primary astroglial cultures depleted of microglia and oligodendrocytes are used to study gliosis in vitro. Astrocyte-enriched cultures can be subjected to focal mechanical injury by a scraping of a cell layer or once cultured on deformable SILASTIC membranes subjected to rapid, reversible stretch-induced injury. Inﬂammatory conditions can be imitated by the exposure of rodent primary astrocyte cultures to a complete cytokine mix (CCM) consisting of TNF-a, IL-1b, and IFN-c, which results in an activated state associated with the release of inﬂammatory mediators (Falsig et al., 2006). This model recapitulates many aspects of a reactive astrocytosis, such as astrocytic hypertophy, upregulation of GFAP 1906 KAMINSKA ET AL. Fig. 3. MAPK signaling in cytokine-stimulated hypertrophic astrocytes. Primary astrocytic cultures were isolated as previously described (Zawadzka and Kaminska, 2003) and labeled in green with anti-GFAP-FITC. Astrocytes were activated by an addition of IL-1b, TNFa, and IFNc (each 100 ng/mL). Cytokine-stimulated astrocytes become hypertrophic (A). An addition of the cytokine cocktail strongly increased the levels of phophorylated MAPK (B) and stimulated the NF-jB transcriptional activity in cells transiently transfected with the luciferase reporter gene under control of the 5 NF-jB binding sites (C). (Fig. 3A), and chemokine and cytokine production, and has been used by us and others to study astrocytic cytokine signaling. Figure 3B,C shows a rapid activation of MAP kinases and upregulation of NF-jB-driven transcription, which leads to the production of inﬂammation mediators. presence of a common intracellular Toll-IL-1 receptor (TIR) domain. Adaptor proteins containing TIR domain can be differentially recruited to the Toll-IL-1 receptors and determine the speciﬁcity of signaling. These adaptors, include MyD88 (myeloid differentiation factor 88); TIRAP (TIR-domain-containing adaptor protein); TRIF (TIR-domain-containing adaptor protein inducing IFNb); and TRAM (TRIF-related adaptor molecule). Activation of TLR4 triggers two major intracellular pathways, the MyD88-dependent and MyD88-independent, TRAM-dependent pathways. The MyD88-dependent pathway mainly induces proinﬂammatory cytokines such as TNFa, IL-6 through activation of MAPK and NF-jB. The TRAM-dependent pathway predominantly induces type I interferons and chemokines such as IP-10 and interferon (IFN)-induced proteins through activation of interferon regulatory factor (IRF) 3 or 7 and NF-jB (Yamamoto et al., 2003). Both pathways complement each other in the production of proinﬂammatory mediators. Genetic and biochemical studies revealed that while most TLR and IL-1 receptor use the adaptor molecule MyD88 to mediate inﬂammatory signal transduction, double-stranded RNA triggered, TLR3-mediated signaling is independent of MyD88 (Akira et al., 2006). MAPK SIGNAL TRANSDUCTION IS A KEY MEDIATOR OF MICROGLIA ACTIVATION AND THE PRODUCTION OF INFLAMMATION MEDIATORS Signaling via Toll-TNF-Interleukin-1 Receptor Superfamily In response to brain injury or infection, intracellular signaling pathways are activated in microglia to carry the signal needed to perform effector inﬂammatory and APC functions. Primary inﬂammatory stimuli (microbial products, protein aggregates, unfolded proteins) and cytokines such as IL-1b and TNFa act through the Toll receptors, IL-1 receptor (TIR) family or the TNF receptor family, respectively (Olson and Miller, 2004). The Tollinterleukin-1 receptor superfamily is deﬁned by the MAPK SIGNALING IN NEUROINFLAMMATION The stimulation with LPS activates multiple TIR-domain-containing adaptors and induces pathways leading to both NF-jB and IRF3 activation (Li and Qin, 2005). TLR signaling results in the downstream activation of three major families of proteins important in the activation of inﬂammatory gene expression: MAPK, nuclear factor-jB (NF-jB)/Rel proteins and IFN regulatory factors. The MyD88-dependent pathway, which is used by most TLRs, recruits IL-1R-associated kinase (IRAK)-1 and IRAK-4 and TNF receptor-associated factor 6 (TRAF6), which afterward dissociates from the receptor complex and associates with another complex composed of transforming growth factor b-activated kinase (TAK1) and TAK1-binding protein 1. This complex formation leads to the activation of TAK1, which in turn activates the transcription factors NF-jB and activator protein 1 (AP-1) through the canonical IjB kinase (IKK) complex and the mitogen-activated protein kinase pathway, respectively (Li and Qin, 2005; Akira et al., 2006; Hu et al., 2007; Uematsu and Akira, 2007). A growing body of evidence demonstrates that activation through TLRs is involved in neurodegeneration (Tang et al., 2007). TLR2-deﬁcient mice develop decreased CNS injury when compared with wild-type mice after focal cerebral ischemia (Lehnardt et al., 2007). TLR-4 deﬁciency protects mice against ischemia and axotomy-induced retinal ganglion cell degeneration (Kilic et al., 2008). TNFa acts through two cell surface receptors: TNFR1 and TNFR2. Activation of TNFR1 leads to the recruitment of the adaptor TRADD (TNFR-associated DD), which can further recruit receptor-interacting protein kinase 1 (RIP1) and TNFR-associated factors (TRAFs), which trigger NF-jB activation, leading to cell survival and proinﬂammatory responses. Recent studies demonstrated involvement of TRADD also in the TLR4 complex formation after LPS stimulation. TRADD-deﬁcient macrophages show impaired cytokine production in response to LPS and poly (I:C) in vitro (Chen et al., 2008; Ermolaeva et al., 2008). TRAF6 has been implicated in mediating the signals from members of different receptor families, including the TNF receptor superfamily, IL-1R/ Toll-like receptor superfamily (IL-1R/TLRSF), TGFbR, IL-17R, and IL-25R, and the NOD-like pattern recognition receptors. TRAF6 activation leads to downstream activation of PI3K, MAPK cascade, and the transcription factors NF-jB, NFAT, and IRF (Aggarwal, 2003; Wu and Arron, 2003). Activation of NF-jB by the proinﬂammatory cytokines and other stress-like stimuli requires the IKK complex, which contains the kinases IKKa, IKKb, or IKKc and the regulatory NEMO (NF-jB essential modulator) subunit (Li et al., 2002). Although IL-1b and TNFa both require NEMO for classical NF-jB activation, NEMO forms a functional IKK complex with IKKa in response to IL-1b, but TNFa requires IKKb to form a signaling unit (Solt et al., 2007). Spatial and temporal separation of IKK and MAPK signaling cascades determine biological speciﬁcity and functional outcome in TNFR signaling (reviewed in Bonizzi and Karin, 2004; Karin and Gallagher, 2009). Lys48-linked ubiquitination of TRAF3 marks it for degradation by the proteasome that releases the brake on TRAF2/6:MAP3K (the MAPK kinase kinase) complexes, leading to their translocation from the cytoplasmic part 1907 of the receptor to the cytosol where they initiate MAPK phosphorylation and activation (Liao et al., 2004). The relevant MAP3Ks and downstream kinases are illustrated in Fig. 4. TRAFs induce the phosphorylation of MAP3K: MEKK1 at Thr1381 and TAK1 at Thr187 and Thr184 within their activation loops (Matsuzawa et al., 2008). MEKK1 phosphorylates MKKs 3, 4, 6, and 7, the MAP2Ks that are responsible for JNK and p38 activation. This phosphorylation occurs within a matter of several minutes. More recently, gene ablation and short hairpin RNA approaches have been used to identify and verify several physiologically relevant MAP3Ks involved in JNK and p38 activation by TNFR family members (Karin and Gallagher, 2009). IKK activation proceeds faster than MAPK activation, takes place at the receptor, and is independent of TRAF3 degradation. MEKK1 is a major contributor to activation of JNK and p38 by two B cell TNFR family members: CD40 and BAFF receptor. CD40 forms a complex containing adaptor molecules TRAF2, Ubc13, and IKKc, which are required for the activation of MEKK1 and downstream MAPK cascade (Matsuzawa et al., 2008). Signaling via MAP Kinase Cascades Three major groups of distinctly regulated MAP kinase cascades that lead to altered gene expression: ERK1/2, JNK, and p38 MAP kinase are known in humans. ERK1/2 is activated by MKK1 and MKK2, JNK by MKK4 and MKK7, and p38 MAP kinase by MKK3, MKK4, and MKK6, respectively. Upon activation of the MAP kinases, transcription factors present in the cytoplasm or nucleus are phosphorylated and activated, leading to expression of target genes resulting in a biological response (Fig. 4). The multiple interactions between the different MAP kinase cascades serve to integrate the responses and activate separate sets of genes (Pearson et al., 2001; Karin and Gallagher, 2009). Termination of signal transduction and negative regulation of MAPK activity is executed primarily by MAPK phosphatases (MKPs), a group of 11 dual-speciﬁcity phosphatases (known also as DUSP) which dephosphorylate the MAPKs on their regulatory threonine and tyrosine residues. MKP-1 localizes to the nucleus and preferentially dephosphorylates activated p38 MAPK and JNK. Lack of MKP-1 affects the production of both proinﬂammatory and anti-inﬂammatory cytokines after LPS treatment (Chi et al., 2006). MAPK Signal Transduction Is a Key Mediator of the Production of Inﬂammation Mediators The expression of proinﬂammatory cytokines can be regulated at both the transcriptional and post-transcriptional levels (Kaminska, 2005; Clark et al., 2009). MAP kinases are key mediators of eukaryotic transcriptional responses to extracellular signals and control gene expression via the phosphorylation and regulation of transcription factors, coregulatory proteins and chromatin proteins (Whitmarsh, 2007). The most prominent example is phoshorylation of c-Jun and ATF-2 proteins by JNK and p38 MAPK leading to increased transcriptional activity of the AP-1 transcription factor. ETS-domain ternary complex factors (TCFs), including Elk-1 and SAP-1, are phosphorylated and activated by ERK, 1908 KAMINSKA ET AL. Fig. 4. The mitogen-activated protein (MAP) kinase signal transduction pathway and its pharmacological modulation. MAPK family is composed of three modules serially activated kinases: a MAPK kinase kinase (MAP3K), a MAPK kinase and ERK, JNK and p38 MAPK. Several substrates or indirect targets of each pathway and their speciﬁc inhibitors are indicated. Abbreviations: ATF, activating transcription factor; ASK, apoptosis signal-regulating kinase; CBP, CREB binding protein; CREB, cAMP response element binding protein; Elk-1, Etslike transcription factor; ERK, extracellular signal-regulated kinase; Ets, transcription factor with v-ets domain (viral-E-26); GADD 153, growth arrest and DNA damage-inducible protein 153; iNOS, inducible nitric oxide synthase; JNK, c-Jun amino-terminal kinase; MAPKAP-K2, MAP kinase-activated protein kinase 2; MEF, myocyte-enhancing factor; MEK, MAP/ERK kinase; MEKK, MEK kinase; MLK, mixed-lineage protein kinase; MyD88, myeloid differentiation factor 88; NFAT4, nuclear factor of activated T cells 4; NIK, NF-jB-inducing kinase; PAK, p21-activated kinase; TAK, transforming growth factor-b-activated protein kinase; TAO, one thousand and one amino acids; TGF-b, transforming growth factor b; TLR, Toll-like receptors; TBP, TATA box binding proteins; TNF, tumor necrosis factor; Tpl-2, tumor progression locus 2 kinase; TRAF, TNF receptor-associated factor; TRAM, TRIFrelated adaptor molecule; TRIF, Toll/IL-1R domain-containing adaptorinducing IFN-beta. JNK, and p38, or ERK and p38, respectively, leading to enhanced transcription of the c-fos gene (Yang et al., 2003). Upregulation of immediate early genes such as cjun and c-fos followed by the formation of the AP-1 transcription factor is required to target promoter elements of many genes (Whitmarsh, 2007). In many cases, coordination of multiple MAP kinase pathways is required to upregulate cytokine expression. For example, a strong induction of TNF promoter-driven gene expression was observed when all of the four MAP kinase pathways were activated simultaneously, suggesting a cooperative effect among these kinases (Zhu et al., 2000). Besides phosphorylation of transcription factors in the nucleus, MAPK are involved in the regulation of several kinases regulating the protein translation. ERK and p38 MAPK phosphorylate MAPK signal-integrating kinase 1 (Mnk1), which phosphorylates an eukaryotic initiation factor 4E (eIF4E), crucial for the initiation of protein MAPK SIGNALING IN NEUROINFLAMMATION synthesis. The p38 pathway has been implicated in the regulation of mRNA stability of cyclooxygenase 2, TNFa, IL-3, IL-6, IL-8, MCP-1a, granulocyte-macrophage colony-stimulating factor (GM-CSF), vascular endothelial growth factor, urokinase-type plasminogen activator, and inducible NO synthase genes (Clark et al., 2003). The effect of p38 MAPK on TNF-a, IL-6, and IFNc synthesis is mainly mediated via the activation of MAPKAP-K2 (mitogen-activated protein kinase-activated protein kinase 2/MK2), because the LPS-induced production of these cytokines is decreased by 90% in MK2-deﬁcient mice (Kotlyarov et al., 1999; Neininger et al., 2002). p38 MAPK a/b activates the nuclear MK2, and as a result of this activation MK2 is exported from the nucleus to the cytoplasm. Active MK2, in turn leads to the phosphorylation of its substrates, such as small heat shock protein Hsp25/27, tyrosine hydroxylase, leukocyte-speciﬁc protein 1, and tristetraprolin (TTP). TTP is the only transacting factor shown to be capable of regulating adenine/ uridine-rich element (ARE)-dependent mRNA turnover (Brooks et al., 2004). Recent studies using MK2- and TTP-deﬁcient mice demonstrated that TTP is the major target of MK2 involved in the post-transcriptional regulation of TNF. TTP binds ARE in the 30 -untranslated region of TNF mRNA and destabilizes TNF mRNA. MK2 activity leads to stabilization of TTP mRNA and increased TTP protein stability but reduced afﬁnity to ARE (Hitti et al., 2006). INHIBITORS OF p38 MAPK AND JNK SIGNALING PATHWAYS AS POTENTIAL NEUROPROTECTIVE DRUGS Distinct Classes of Inhibitors of MAPK Signaling Pathways Since the discovery of p38 MAPK involvement in the synthesis of IL-1b and TNFa in 1994 (Lee et al., 1994), it was well established that the main biological consequence of p38 activation is the production of inﬂammatory mediators to initiate leukocyte recruitment and activation. To date, four p38 MAP kinase isoforms have been identiﬁed sharing about 60% homology. Two isoforms (p38a and p38b) are ubiquitously expressed, p38c is predominantly expressed in skeletal muscle, whereas p38d gene expression is found in the lung, kidney, testis, pancreas, and small intestine. p38a is the major isoform of p38 MAPK, activated under inﬂammatory conditions and crucial for the production of inﬂammation mediators. p38 MAPK is activated by the dual phosphorylation on Thr180 and Tyr182 by upstream MAPK kinases: MKK3 and 6, which are activated by upstream MAP3Ks. Extracellular signals activating the p38 MAP kinase pathway include a variety of cytokines (IL-1a, IL-2, IL-7, IL-17, IL-18, TGF-b, and TNF-a) and pathogenic stimuli, including LPS, staphylococcal peptidoglycan and enterotoxin B, echovirus 1, and herpes simplex virus 1 (Olson and Miller, 2004). MAPK inhibitors emerge as attractive anti-inﬂammatory drugs, because they are capable of reducing both the synthesis of inﬂammation mediators at multiple levels by transcriptional, post-transcriptional, and translational repression and are effective in blocking proinﬂammatory cytokine signaling. Following the pioneering work with p38 MAPK inhibitors such as SB 1909 203580 (developed by SmithKline Beecham), a vast number of inhibitors of MAPK (Fig. 4) has been characterized in vitro and in animal models, and several compounds have been advanced into clinical trials. The commonly used inhibitor SB 203580 is known to inhibit both p38a and p38b isoforms by binding to the ATP pocket of p38 MAPK. A second generation of p38 MAPK inhibitors shows a better selectivity. SB 239063 exhibits the equipotent inhibitory activity against the aand b-isoforms and no activity (up to 100 lM) against the c- and r-kinase isoforms (reviewed in Kaminska and Swiatek-Machado, 2008). Another class of inhibitors is represented by BIRB 796 BS [1-(5-tert-butyl-2-p-tolyl2H-pyrazol-3-yl)-3-[4-(2-morpholin-4-yl-ethoxy)-naphtalen1-yl]-urea], a water-soluble molecule developed by Boehringer Ingelheim Pharmaceuticals. In contrast to other p38 MAPK inhibitors (e.g., SB 203580), BIRB 796 BS prevents both the phosphorylation and activity of p38 MAPK by binding to a novel allosteric binding site as well as to the ATP pocket of p38 MAPK. The studies in humans demonstrated that the orally administered BIRB 796 BS signiﬁcantly inhibited LPS-induced p38 MAPK activation in the leukocyte fraction, as well as cytokine production (Branger et al., 2002). Semapimod (formerly known as CNI-1493) is a synthetic guanylhydrazone (developed by Cytokine PharmaSciences), which inhibits MAPK signal transduction pathway by preventing phosphorylation of MEK, p38 MAP kinase, and JNK via yet unknown mechanism and affects the production of the proinﬂammatory cytokines TNFa, IL-1b, IL-6, MIP-1 a/b, and NO (Bianchi et al., 1995; Cohen et al., 1996). Semapimod has been shown to inhibit macrophage activation and to impair maturation of dendritic cells associated with a reduction of DC-maturation marker (CD83) (Zinser et al., 2004). CEP-1347 (from Cephalon) is a potent inhibitor of mixed lineage kinases (MLKs), a distinct family of mitogen-activated protein kinase kinase kinases (MAP3K), which acts upstream of p38 and JNK activation in macrophages (Maroney et al., 2001). CEP-1347 inhibited MAPK-mediated neuronal death in vitro and in animal models of neurodegenerative diseases. CEP-11004, a new MLK inhibitor, efﬁciently inhibited TNF-a production in LPS-stimulated THP-1 cells and BV-2 microglial cells, as well as in mice when injected 2 hr prior to LPS administration (Ciallella et al., 2005). CEP-1347 and CEP-11004 blocked the activation of murine astrocytes by either TNF plus IL-1 or by a CCM containing IFN-c. The compounds blocked production of NO, prostaglandin E2, and IL-6 with a median inhibitory concentration (IC50) of approximately 100 nM. Although CEP-1347 did not affect the nuclear translocation of NF-jB, it blocked the expression of Cox2, iNOS, TNF, GM-CSF, urokinase-type plasminogen activator, and IL-6 at the transcriptional level (Falsig et al., 2004). SP600125, an anthrapyrazolone inhibitor of JNK (developed by Celgene), is a reversible ATP-competitive inhibitor. SP600125 effectively inhibited the phosphorylation of c-Jun, the production of IFNc, TNFa, and IL-10 and weakly affected IL-1b and IL-6 production in Th1 cells. In Th2 cells, SP600125 potently blocked TNFa and IL-10 but had no effect on IL-4. In human peripheral blood monocytes stimulated with LPS, SP600125 reduced elevation of COX-2 and TNFa mRNA in a dose- 1910 KAMINSKA ET AL. dependent manner, while it had no effect on the levels of ICAM-1, IL-1b, and IL-8 mRNA (Bennett et al., 2001). PD098059 (20 -amino-30 -methoxyﬂavone), a MEK1 inhibitor, and UO126, 1,4-diamino-2,3-dicyano-1,4-bis(2aminophenylthio)butadiene, a dual MEK1 and MEK2 inhibitor, are selective inhibitors of ERK signaling pathway (Fig. 4). ERK signaling is activated by a wide variety of external signals leading to cell proliferation or differentiation and cell survival (Pearson et al., 2001). Inhibitors of p38 MAPK and JNK Signaling Pathways as Neuroprotective Drugs Inhibitors of p38 MAPK (e.g., SB 203580, RWJ 67657, BIRB 796 BS) have been found to reduce LPS-induced cytokine production in mice during systemic endotoxemia (Badger et al., 1996; Wadsworth et al., 1999) and in the leukocyte fraction in human volunteers (Branger et al., 2002). Sustained activation of p38a MAPK was observed in activated microglia after cerebral brain ischemia, and its inhibitor SB 203580 signiﬁcantly reduced the infarct size in the gerbil global ischemia model (Sugino et al., 2000). A second-generation p38 MAPK inhibitor, SB 239063, decreased the infarct volume and neurological deﬁcit in rats after transient (Legos et al., 2001) and permanent (Barone et al., 2001) MCAO. SB 203580, delivered by the intraventricular injection 30 min before, 6 or 12 hr after MCAO, reduced the infarct volume and improved neurological deﬁcits. SB 203580 attenuated the expression of iNOS, TNFa, IL-1b, and Cox-2 when administrated 6 hr after MCAO (Piao et al., 2003). Transgenic mice overexpressing APP751 (a transgenic mouse model of Alzheimer’s disease) have increased inﬂammatory responses and showed upregulation of phosphorylation of p38 MAPK. Ab exposure has been shown to stimulate p38 MAPK phosphorylation in cultured microglia (McDonald et al., 1998). SD282, an inhibitor of p38 MAPK, reduced activation of microglia and protected the APP751-overexpressing mice against ischemic injury (Koistinaho et al., 2002). The MLK inhibitor CEP-1347 blocked the activation of the c-Jun/JNK signaling pathway in neurons exposed to stress and attenuated neurodegeneration in animal models of Parkinson’s disease (Saporito et al., 1999). The studies by Lund et al. (2005) demonstrated that CEP1347 reduces cytokine production in primary cultures of human or murine microglia and in monocyte/macrophage-derived cell lines, stimulated with endotoxins or Ab1-40. Moreover, CEP-1347 inhibited TNF production in the brain induced by intracerebroventricular injection of LPS in mice. In primary microglial cultures, SP600125 (1–5 lM) JNK inhibitor reduced the LPS-induced metabolic activity and upregulation of Cox-2, TNF-a, MCP-1, and IL-6, while inhibition of ERK1/2 by 40 lM PD98059 and p38a by 5 lM SB 203580 had a more pronounced effect on LPS-induced cellular enlargement. Ciclosporin A (CsA, formerly cyclosporin A), FK506 (tacrolimus), and SDZ ASM 981 (pimecrolimus) are short peptides used to block graft rejection in transplantation of organs and bone marrow. They bind to speciﬁc intracellular proteins called immunophilins and such complexes inhibit calcineurin (PP2B, PP3), a calciumand calmodulin-dependent threonine/serine phosphatase. FK506 and CsA exert the neuroprotective action in animal models of neurologic diseases: traumatic brain injury, spinal cord injury, optic nerve crush, antiretroviral toxic neuropathy, rodent models of Parkinson’s disease, and stroke (reviewed in Guo et al., 2001; Kaminska et al., 2004). We demonstrated that FK506 (1 mg/kg, i.v.) injection 1 hr after MCA occlusion greatly reduces the activation of microglia and astrocyte in the injured cortex, the infarct volume, and neurologic deﬁcits. FK506 inhibited upregulation of IL-1b and TNFa mRNA expression in the rat ischemic brain and in LPSstimulated microglial cultures (Zawadzka and Kaminska, 2005). Although immunosuppressants are not direct MAPK inhibitors, our studies demonstrate that FK506 strongly inhibits LPS-induced activation of p38 MAPK and JNK in microglial cells (unpublished). In addition, FK506 reduced astrocyte proliferation and the levels of TNFa and IL-1b mRNAs in cultured cortical astrocytes under basal conditions (Zawadzka and Kaminska, 2005) and impaired astrocytic hypertrophy in the cytokinecocktail-stimulated astrocytes and reduced H2O2-induced activation of MAPK in cell culture models of astrogliosis (Gozdz et al., unpublished). Altogether, our results demonstrate the ability of FK506 to block an early activation of glial cells and brain inﬂammation, probably via interference with MAPK signaling. CONCLUSIONS Although brain inﬂammation and gliosis are hallmarks of brain pathology and targets of anti-inﬂammatory drugs, the appreciation of the beneﬁcial side of neuroinﬂammation (Schwartz et al., 2009) has initiated the evaluation of costs and beneﬁts of this process in animal models of neurological disorders. Recent studies using animals with genetic or pharmacological inactivation of microglia/macrophages point to a critical role of the timing and the extent of microglia activation, distinct roles of different populations (resident versus CNSinﬁltrating macrophages) at different time points following injury. The in vivo function of microglia and the extent of contribution to the onset, progression, and recovery from chronic inﬂammation-linked disorders are still a matter of debate, partly due to the contrasting results derived from the different models used to assess microglial function (Carson et al., 2008). Resident microglial cells are involved in the removal of damaged, unwanted cells and participate in repair processes, so the extent of inﬂammatory microglia activation would determine whether detrimental effects outweigh neuroprotective mechanisms. Understanding of signaling pathways underlying the initiatory phase of microglia activation, synthesis of proinﬂammatory cytokines and neurotoxic molecules will facilitate ﬁne-tuning of microglial function and development of novel diagnostic and therapeutic approaches. Understanding of signal transduction involved in microglia-mediated inﬂammation allows the discovery of compounds which can be useful in the therapy of neurological disorders. The inhibitors of p38 MAPK and JNK signaling pathways are clearly attractive because they are capable of reducing both the synthesis of proinﬂammatory cytokines and their intracellular signaling. Second-generation inhibitors targeting activity of p38 MAPK have been improved with respect to drug MAPK SIGNALING IN NEUROINFLAMMATION speciﬁcity. FK506, interfering with MAPK signaling, represents another class of promising drugs with wellknown pharmacokinetics and safety. 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