Signaling Transduction

An essential feature of the early immune response to pathogens and cell debris is secretion of proinflammatory cytokines. So, understanding how cytokine signaling works can provide strategies to look for new therapeutic targets in several inflammatory and autoimmune disorders and also to improve mechanisms of defense against several pathogens. In a simplistic description, both TNF-a and IL-ip signaling activate similar pathways which culminate with the increased expression of target genes, most of which encode proteins involved in immunity and inflammation. Both IL-ip and TNF-a receptors lack metabolic or enzymatic activity and recruit several intracellular adaptor proteins responsible for transducing the signal downstream receptor level, amplifying a cascade of signal transduction pathways for both cytokines (MacEwan, 2002).

It is important to note that the majority of experimental work aiming to unravel the signal pathways activated by these two cytokines has been done in diverse experimental models including blood cells and different cell lines. The obtained data lead people to think that similar mechanisms are responsible for most IL-ip and TNF-a actions in the CNS. However, we must keep in mind that similar pathways, in different experimental models, do not necessarily result in the same toxic or protective effects partially because some signaling proteins and their interactions can slightly differ.

5.1. IL-1P Signaling

IL-ip is a pleiotropic cytokine that has been implicated in a number of neurodegenerative/neurotoxic conditions and is generally assumed to result in neurotoxic processes (Bernardino et al., 2005a, b), although it has been also reported that IL-ip can play a neuroprotective role (Rothwell and Luheshi, 2000; Friedman, 2005; Sayyah et al., 2005). IL-i p can be expressed by astrocytes, oligodendrocytes, neurons, microglia, cerebrovascular cells and circulating immune cells invading the CNS upon injury (Davies et al., i999; Pearson et al., i999). The inactive 3i kDa IL-ip is cleaved by a highly specialized IL-ip converting enzyme (ICE or caspase-i) into a i7 kDa IL-ip mature protein (Thornberry et al., i992). The IL-i family consists of the two agonists IL-ia and IL-ip and the endogenous IL-i receptor antagonist (IL-ira), all of which bind to specific IL-i receptors such as type I (IL-iRI) and type II (IL-iRII) receptors. The ligands bind both receptors with distinct affinities (Dinarello, i996). IL-iRI mediates the biological effects of IL-ip (Sims et al., i988, i993; Dinarello, i996), whereas IL-iRII is not capable of transducing the IL-ip mediated signal, but rather acts as a decoy receptor, competing with the IL-iRI for IL-ip (Colotta et al., i993). This is mainly due to the fact that IL-iRII contains only a short cytoplasmic tail and do not contain a TIR domain (the effector region of the IL-iRI receptor). On the other hand, the endogenous IL-ip antagonist, IL-ira, also binds to IL-iRI. So, IL-iRII and IL-ira can act together and cooperate in order to regulate the interaction between IL-ip and its receptor IL-iRI.

Another member of the IL-i receptor family, IL-i receptor accessory protein (IL-iRAcP), was also identified. The IL-iRAcP shares limited homology with IL-iRI and IL-iRII and does not bind IL-ip, but appears to increase the affinity of II-iRI for IL-ip (Greenfeder et al., i995). It has been shown that co-expression of the IL-iRAcP is essential for a fully functional IL-iRI complex (Greenfeder et al., i995; Hofmeister et al., i997).

In rat and mouse brain, distribution of the II-1RI was initially investigated using radiolabeled IL-1a and p. These studies showed high density of binding sites particularly in the granular cell layer of the dentate gyrus of the hippocampus and a weak to moderate signal was obtained over the pyramidal cell layer of the hylus and the CA3 subfield. The signal was also detected in choroids plexus, meninges, and anterior pituitary (Takao et al., 1990; Ban, 1994; Loddick et al., 1998).

IL-1RI has been recognized as part of an interleukin-1 receptor/Toll-like receptor (IL-1R/TLR) superfamily, whose members are involved in host defense and inflammation (Gay and Keith, 1991; O'Neill and Greene, 1998). Moreover, these two receptors share sequence similarity in their cytosolic regions, the TIR domain, and, accordingly, they have similar signaling pathways (Li and Qin, 2005).

Following IL-1RI and IL-1RAcP complex formation induced by IL-1p (Greenfeder et al., 1995; Wesche et al., 1997), the intracellular adaptor protein, termed Myeloid Differentiation factor 88 (MyD88), is recruited and interact with the carboxyl-terminal TIR domain of the IL-1RI (Kawai et al., 1999; Li et al., 2005; Davis et al., 2006). Then, the amplification of signal requires the interaction between IL-1RI/MyD88 and an IL-1 Receptor-Associated Kinase (IRAK). Recruitment of IRAK to IL-1RI/MyD88 complex is mediated by a scaffolding protein, called Toll-interacting protein (Tollip) (Burns et al., 2000). In resting cells, Tollip forms a complex with IRAK and inhibits IL-1RI signaling by binding to and blocking IRAK phosphorylation. Upon activation, Tollip-IRAK complexes are recruited to the activated receptor, allowing the interaction between IRAK and MyD88 (Croston et al., 1995; Cao et al., 1996a; Zhang and Ghosh, 2002). IRAK then undergoes rapid auto-phosphorylation and, in turn, phosphorylates Tollip, resulting in the dissociation of IRAK-Tollip complexes and the activation of downstream signaling components (Burns et al., 2000). IRAK becomes hyperphosphorylated, leaves the receptor complex and interacts with Tumor necrosis factor Receptor-Associated Factor 6 (TRAF6) (Cao et al., 1996b; Yamin and Miller, 1997; Anderson, 2000). TRAF6 then activates NFkB inducing kinase (NIK) (Baeuerle, 1998), a mitogen activated protein kinase kinase kinase (MAPKKK), which in turn phosphorylates and activates the IkB kinase (IKK) complex (Fig. 1). Alternatively, another MAP kinase kinase kinase named TAK1, in association with TAK1-binding proteins 1 and 2 (TAB1 and TAB2), has been identified to interact with TRAF6 (Takaesu et al., 2000, 2001; Qian et al., 2001; Kishida et al., 2005), thereby activating also the IKK complex, leading to NFkB activation (DiDonato et al., 1997; Mercurio et al., 1997; Zandi et al., 1997; Meffert and Baltimore, 2005). Although, it remains unclear which members of the TAK1/TAB1/TAB2 complex are essential for IL-1p signaling (Shim et al., 2005).

In resting cells, NFkB proteins are present in the cytosol as heterodimer of proteins of the Rel family of transcription factors associated with proteins that are known as inhibitory proteins of NFkB (IkBs) (Thanos and Maniatis, 1995; Baeuerle and Baltimore, 1988). The IkBs proteins include IkBs, IKBa, IkBP, which trap NFkB dimers in the cytoplasm by masking its nuclear localization signal (NLS) and by this process prevent NFkB binding to DNA. In response to a stimulus, such as IL-1RI activation, the degradation of the inhibitory IkBs proteins involves phosphorylation mediated by IkB kinases (IKK) complex. IKK complex is composed by two catalytic subunits, IKKa and IKKp, and one regulatory subunit, NEMO (IKKy). Both the IKKy/NEMO and IKKa subunits are required for activation of NFkB by stimuli such as IL-1p and TNF-a, although only the IKKp catalytic subunit is essential to trigger NFkB activation (Verma et al., 1995; Rivest, 2003). So, the IKKp subunit is activated by NIK and phosphorylates IkB proteins leading to the ubiquitin-dependent degradation of IkB by the 26S proteasome subunit (Traenckner et al., 1994, 1995; Delhase et al., 1999). Then, NFkB, free of IkB proteins, translocate to the nucleus and binds to DNA binding sites regulating the transcription of several genes, including inducible cyclo-oxygenase (which leads to the production of prostaglandins, important inflammatory mediators), inducible form of nitric oxide synthase, adhesion molecules, chemokines, cytokines, immune receptors, and growth factors (Moynagh et al., 1994) (Fig. 1). Following its degradation, IkB proteins are rapidly re-synthesized to act as endogenous inhibitors of NFkB.

Besides the NFkB pathway, the recruitment of MyD88/IRAK/TRAF6 complex is also able to activate MAPK kinases (MKKs), in particular the JNK pathway that leads to AP-1 activation (Rivest, 2003) and also the p38 MAP kinases (O'Neill, 2002; Dunne and O'Neill, 2003; Wang et al., 2005b). The specific pathways activated by IL-1P may differ in distinct cell types and may mediate distinct biological consequences of IL-1P (Srinivasan et al., 2004).

5.2. TNF-a SIGNALING

TNF-a is a pleiotropic inflammatory cytokine expressed as a 26 kDa transmembrane precursor from which a soluble 17 kDa polypeptide is released after proteolytic cleavage, mainly by the metalloprotease TNF-a converting enzyme (TACE) (Karkkainen et al., 2000). It was reported that TNF-a is active either as a membrane bound or as a soluble form (Kriegler et al., 1988; Idriss and Naismith, 2000). In general, TNF-a, can be produced by activated microglia and astrocytes, but under pathological conditions TNF-a can also be produced by CNS-infiltrating lymphocytes and macrophages (Vassalli, 1992).

TNF-a can interact with two different receptors (TNFR1 and TNFR2) that are transmembrane proteins with an extracellular carboxy-terminal with cysteine-rich domains and an amino-terminal intracellular domain, both linked by a single transmembrane domain. The C-terminal extracellular domain has approximately 28% amino acid identity between both TNF-a receptors and is responsible for the assembly of receptor trimers and their binding properties (Aggarwal, 2003). The extracellular domain of both TNFR1 and TNFR2 can be proteolytically cleaved, giving rise to soluble receptors for TNF (sTNFR), which can neutralize TNF-a (Wallach et al., 1991). Moreover, unlike IL-1/Toll like receptors, TNF-a receptors show almost no homology in their intracellular sequences and suggest that TNFR1 and TNFR2 activate distinct signaling pathways, contributing to a variety of different biological responses mediated by TNF-a (Grell et al., 1994).

TNF-a-induced signal transduction starts with the pre-assembling of both TNFR1 and TNFR2 on the cell membrane before TNF-a binding. The formation of this complex requires the extracellular pre-ligand-binding assembly domain (PLAD), a highly conserved domain containing cysteine resides in the extracellular region of these receptors. PLAD mediates ligand-independent assembly of receptor trimers and facilitates the interaction between TNF-a and its receptors (Chan, 2000; Chan et al., 2000; Jones, 2000; Locksley et al., 2001). Nevertheless, TNF-a signaling is only transduced following physical binding of TNF-a to TNF receptors.

5.2.1. TNFRl-Mediated Signaling

Among the two TNFR, TNFR1 has been the mostly investigated receptor, partially due to its potential role in the control of cell death and consequently they are looked as good potential therapeutic targets in several diseases. The main difference between TNFR1 and TNFR2 is the presence of intracellular regions so-called death domains (DD) in the TNFR1 (Itoh and Nagata, 1993; Tartaglia et al., 1993). DD are also present on other associating proteins that are primarily involved in cell death signaling. Moreover, deletion of DD region abolishes ligand-induced apoptosis, indicating that this region is required for TNF-a-induced apoptosis. Moreover, silencer of death domain (SODD) proteins associate constitutively with DD region of TNFR1 and so inhibits the recruitment of several DD-interacting proteins, and prevent ligand-independent activation of TNFR1 and spontaneous cell death signaling (Jiang et al., 1999; Takada et al., 2003). After a stimulus, TNF-a binds to the TNFR complex and the SODD is released from the DD of TNFR1, allowing the interaction between TNFR1 and adaptor cytoplasmic molecules containing DD domains (MacEwan, 2002).

TRADD-Mediated Pathways

After binding of TNF-a, TNFR1 recruits a cytoplasmic protein called TNFR associated death domain (TRADD) acting as a platform adaptor for the downstream TNF-a signaling. TRADD interacts with the TNFR1 cytoplasmic domain by DD regions in both molecules. This TRADD-DD complex recruits the downstream signaling adaptor molecules fas-associated death domain (FADD) and receptor interacting protein (RIP) (Aggarwal, 2000; Park et al., 2005). FADD is the major adaptor protein involved in the TNFR1 mediated-cell death. Besides a DD at the carboxyl terminus able to interact with TRADD-DD complex, FADD contains a death effector domain (DED) motif allowing the interaction and activation of caspases and other DED-containing molecules involved in execution of cell death (Chinnaiyan et al., 1995; Hsu et al., 1996b; Park et al., 2005). So, FADD can activate caspase-8 known as an apoptotic initiator caspase, that in turn activates the executioner caspases such as caspase-3 and -6, causing cell death (Kischkel et al., 2000; Denecker et al., 2001; Chen and Goeddel, 2002) (Fig. 2). This death pathway mediated by TRADD, FADD, and caspases is dependent on the internalization of activated TNF/TNFR1 complexes (TNF receptosomes) (Higuchi and Aggarwal, 1994). During the endocytic process, TRADD, FADD and caspase-8 form the death-inducing signaling complex, ultimately resulting in apoptosis (Aggarwal, 2000). This endocytic mechanism can also regulate the activation of TNFR1 and contributes to stop TNF-a signaling.

Another adaptor protein able to interact with the DD of TRADD is RIP. Upon TNF-a binding, TNFR1 is translocated to cholesterol- and sphingolipid enriched membrane microdomains, known as lipid rafts, where it associates with TRADD and RIP, forming a signaling complex. The interaction between TNFR1/TRADD and RIP in lipid rafts is a potent inducer of apoptosis (Legler et al., 2003). Moreover, besides the indirect role on apoptosis, RIP can also play a role in gene expression and proliferation (Blonska et al., 2005; Thakar et al., 2006). RIP can activate NFkB, mediating the activation of IKK, and can also activate JNK (Hsu et al., 1996a) (Fig. 2). Deletion of the gene encoding RIP, however, abolished TNF-induced activation of NFkB, but had minimal effect on JNK activation, indicating that RIP plays a central role in the activation of NFkB (Hsu et al., 1996a; Kelliher et al., 1998). The activation of p38 MAPK by TNFR1 is less well understood and involves the recruitment of RIP by TRADD, which in turn recruits mitogen-activated protein kinase kinase (MKK)3 leading to p38 MAPK activation (Wajant et al., 2003).

Besides FADD and RIP, TRADD can also interact with TNF receptor associated factor (TRAF) proteins, specially TRAF2, that plays a central role in NFkB activation (Hsu et al., 1995, 1996a, b). TNFR1 indirectly interacts with TRAF2 by using the TRADD protein (Fig. 2). TRAF proteins can be involved in death receptor signaling with the indirect recruitment of cytoplasmic adaptor proteins. However, the predominant role attributed to TRAF2 is cell survival through the NIK protein. As previously mentioned in the IL-1ß signaling section, NIK is a member of the serine/threonine mitogen-activated protein kinase kinase kinase (MAPKKK) (Malinin et al., 1997) and has been implicated in TNF-a induced NFkB activation by the phosphorylation of IKBa mediated by the IKK complex (Ling et al., 1998). TNFR1 can also activate JNK through the sequential recruitment of TRAF2, MAP/ERK kinase kinase 1 (MEKK1) and MAPK kinase 7 (MKK7). Deletion of the TRAF2 gene has a minimal effect on TNF-induced NFkB activation, but results in deficient JNK signaling

Tnfr1 Plad

Fig. 1. Schematic representation of the major signal transduction pathways activated by IL-ip. Abbreviations: IL-1R1, IL-1 receptor type I; IL-iRAcP, IL-1 receptor accessory protein; MyD88, myeloid differentiation factor 88; IRAK, IL-1 receptor-associated kinase; Tollip, Toll-interacting protein; TRAF6, Tumor necrosis factor Receptor-Associated Factor 6; NIK, NFkB inducing kinase; IKK, inhibitors of kB (IkB) kinases. Further details are described in the text (see Sect. 5.1) and in the supplementary movies accompanying this book

(Yeh et al., 1997), indicating that TRAF2 is essential for JNK activation but not absolutely required for NFkB activation. NFkB, JNK, and p38 MAPK ultimately lead to the synthesis of immune and inflammatory proteins with several different functions. The activation of NFkB leads to the expression of inflammatory genes such as vascular adhesion molecule-1 (VCAM-1), M1P-1P, MIP-2, and several other cytokines and chemokines (Ghosh and Karin, 2002). Moreover, several NFKB-regulated proteins able to suppress apoptosis have been identified. These include TRAF2, inhibitor of apoptosis 1 (IAP1) and IAP2, survivin, FAS-associated death domain-like interleukin-1 converting enzyme inhibitory protein (FLIP), decoy receptor (DCR) and Bcl-xl. These proteins inhibit different steps of the apoptotic pathways. For example, DCR sequesters the receptors, whereas FLIP blocks the activation of caspase-8, survivin inhibits caspase-3 activation, IAP inactivate effector caspases, and members of the bcl-2 family inhibit cytochrome c release (Wang et al., 1998) (Fig. 2).

Anti Tnf Tnfr2

Fig. 2. Schematic representation of the major signal transduction pathways activated by the two TNF receptors subtypes (TNFR1 and TNFR2). TNF Receptor 1 triggers different signaling pathways via three different functional domains: the death domain (A and B) and the NSD domain or the ASD domain (C). Instead, TNF receptor 2-mediating signaling transduction pathways are mediated by a TIM domain (D). Further details are described in the text (see Sect. 5.2) and in the supplementary movies accompanying this book

Fig. 2. Schematic representation of the major signal transduction pathways activated by the two TNF receptors subtypes (TNFR1 and TNFR2). TNF Receptor 1 triggers different signaling pathways via three different functional domains: the death domain (A and B) and the NSD domain or the ASD domain (C). Instead, TNF receptor 2-mediating signaling transduction pathways are mediated by a TIM domain (D). Further details are described in the text (see Sect. 5.2) and in the supplementary movies accompanying this book

So, while recruitment of DD containing proteins such as FADD and RIP can lead predominantly to apoptosis or necrosis, recruitment of TRAF2 can lead to the activation of transcription factors such as NFkB and AP-1 and therefore promoting inflammatory reactions, cell survival and differentiation (Wajant et al., 2003). So, TNFR1 can simultaneously activate both apoptotic and antiapoptotic signals (Zheng et al., 2006). The apoptotic/necrotic signals do not require protein synthesis, whereas anti-apoptotic signals involve the activation of several transcription factors, including NFkB (Karin and Lin, 2002). The balance between these two signals is regulated at several levels such as regulation of receptor/ligand expression, soluble decoy receptor expression and regulation of protein synthesis.

Sphingomyelinase-Mediated Pathways

Besides the DD in its intracellular terminal, TNFR1 has also other intracellular adjacent regions named neutral-sphingomyelinase activating domain (NSD) and acidic sphingomyelinase activating domain (ASD). Much interest has also been paid to the ability of TNF to stimulate these SMase enzymes (Kolesnick and Kronke, 1998). The proximal half of TNFR1 was found to be responsible for the stimulation of membrane-associated N-SMase partially via ceramide interconverted from diacylglycerol (DAG) (Wiegmann et al., 1994), but also via the Factor Associated with Neutral sphingomyelinase (FAN) (Neumeyer et al., 2006). FAN stimulates N-SMase directly (Adam-Klages et al., 1996; Neumeyer et al., 2006) and therefore catalyses the degradation of sphingolipids into smaller ceramide-containing molecules (Adam-Klages et al., 1998) (Fig. 2). Ceramide production by FAN can induce proliferation or inflammation involving kinase pathways. By decreasing plasma and mitochondrial membrane stability, ceramide can also trigger apoptosis (Malagarie-Cazenave et al., 2002; Pettus et al., 2002; Colombaioni and Garcia-Gil, 2004).

TNF-a can also activate the phosphatidylcholine-specific phospholipase C (PC-PLC) through the TNFR1/A-SMase complex. Stimulation of PC-PLC activity degrades phospha-tidylcholine into phosphocholine and DAG causing the activation of protein kinase C (PKC) isoforms (MacEwan, 2002), downstream activating NFkB cascade (Fig 2).

5.2.2. TNFR2-Mediated Signaling

The overall function of TNFR2 is largely unknown. The main difference between TNFR1 and TNFR2 signaling came from the fact that TNFR2 lacks an intracellular DD. Nevertheless, TNFR2 contains the so-called TRAF interacting motifs (TIM) in its cytoplasmic domain. Activation of TIM leads to the direct recruitment of TRAF-family members and the subsequent activation of signaling pathways such as NFkB, JNK, extracellular signal-related kinase (ERK), p38 and phosphoinositide 3-kinase (Pi3K) (Liu et al., 1996; Hehlgans and Mannel, 2002; Dempsey et al., 2003) (Fig. 2). This can result in the expression of cytokines, but also in the expression of regulatory proteins with potential antiapoptotic activity, such as, TRAF2, IAP1, and IAP2 (Wang et al., 1998).

Even if TNFR2 cannot directly induce apoptosis, it was shown that it can potentiate the effects mediated by TNFR1. Accordingly, it was described that TNFR2 can induce ubiquitination and proteasomal degradation of TRAF2 proteins and this can potentiate TNFR1-mediated apoptosis (Li et al., 2002). As mentioned before, the variety of cellular responses driven by different receptors can be due to the different signaling pathways activated by each receptor or by the expression patterns of the two receptors as well as associated adaptor proteins in different cell types and tissues (MacEwan, 2002; Thommesen and Laegreid, 2005). Moreover, different affinity of both receptors for the soluble or membrane associated TNF-a forms and changes in TNFR1:TNFR2 ratio at the plasma membrane work as mechanisms to control the functional outcome of TNF-a stimulus (Grell et al., 1995). Moreover, experimental conditions in in vitro studies such as the biological model used, the methodology or protocol used and the local concentrations of cytokines can also determine the fate of the cells stimulated with TNF-a. Moreover, TNFR1 has broad functional effects due to its ubiquitous expression in different cell types, while TNFR2 is involved in inflammation since it is expressed mainly by immune and endothelial cells (Aggarwal, 2000).

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