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Nuclear factor kappa B—molecular biomedicine: the next generation
Biomedicine & Pharmacotherapy 58 (2004) 365–371 www.elsevier.com/locate/biopha
Review
Nuclear factor kappa B—molecular biomedicine: the next generation Peter Celec a,b,* a
b
Institute of Pathophysiology, Faculty of Medicine, Comenius University, Bratislava, Slovakia Departmant of Molecular Biology, Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia Received 20 November 2003; accepted 24 December 2003 Available online 08 June 2004
Abstract Nuclear factor kappa B (NFjB) as a transcription factor plays an important integrating role in the intracellular regulation of immune response, inflammation and cell cycle regulation. Nouvelle insights into the structure and regulation of activation of NFjB have brought a detailed picture of the function of this transcription factor. In this review the findings of interactions of NFjB with its inhibitors, tumour necrosis factor alpha and glucocorticoids are presented. The results from the latest in vivo studies show the capability of specific NFjB inhibitors in the clinical use. This article summarizes the most important facts regarding NFjB participation in the pathogenesis of diseases and its potential as a target of pharmacological agents. © 2004 Elsevier SAS. All rights reserved. Keywords: Nuclear factor kappa B; Apoptosis; Cell cycle regulation; Immunosuppressive therapy; Inflammation
1. Introduction
2. The molecules and their regulation
In 1986 Sen and Baltimore [65] first described a nuclear protein that should capture the interest of researchers for the next decades. However, the opinion that this protein is a kappa immunoglobulin enhancer activator specific for mature B cells has changed dramatically. The functional territory has widened extraordinarily. The term nuclear factor kappa B (NFjB) remained untouched. Nowadays, it is clear that NFjB plays a central role in the regulation of cellular processes throughout the tissues. Although the function in immune competent cells is best described, the master position in apoptosis and cell cycle control explains the growing number of publications in molecular biology and biomedical research (Fig. 1). The complex of interactions with other relevant signalling pathways at cellular level and the relation to a number of pathological entities outline the hope that is put to NFjB as a potential pharmaceutical target of the next generation [54].
NFjB is not a single and unique molecule. It is more a functional description for a complex of two variable subunits that come from the Rel/NFjB family. The members (RelA, RelB, c-Rel, p100/p52 and p105/p50) contain a so-called Rel homology domain—a 300 amino acid long sequence that is responsible for the binding to the other subunit and to the
* Corresponding author: Galbaveho 3, 841 01 Bratislava, Slovakia. E-mail address: [email protected] (P. Celec). © 2004 Elsevier SAS. All rights reserved. doi:10.1016/j.biopha.2003.12.015
Fig. 1. Number of publications concerning NFjB per year as the result of a Medline search.
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Fig. 2. A simplified scheme of the activation of NFjB by the degradation of IjB. IjB is phosphorylated by IKK and ubiquinatated by the ubiquitine ligase system (ULS). IjB is further degradated by the 26S proteasome (26S). Activated NFjB can pass the nuclear membrane and interact with jB binding sequences in enhancers of NFjB regulated genes. LPS, lipopolysaccharide; ROS, reactive oxygen species; FasL, Fas ligand; TRAF, TNFa receptor associated factor; NIK, NFjB inducing kinase; MEKK, mitogen activated protein kinase/extracellular signal regulated kinases kinases.
DNA [21]. The best characterized variant is the combination of RelA and p50. Inactivated subunits are localized as homoor hetero-dimers in cytoplasm together with IjB—the NFjB inhibitor. The IjB family has four known members (IjBa, IjBb, IjBe and Bcl3). These proteins contain an ankyrin repeat motif, which is important for the maintenance of NFjB in the cytoplasm [31]. The activation of NFjB lies in the release from IjB (Fig. 2). A great number of stimuli, which are partly mentioned in Table 1, activate IjB kinase (IKK) kinases, which include NFjB inducing kinase (NIK), NFjB activating kinase (NAK), atypical protein kinases C, Akt/protein kinase B, proteins related to IKK, receptor activator for NFjB and its ligand (RANK and RANKL), but also mitogen activated protein kinase kinases (MAPKK), mitogen activated protein kinase/extracellular signal regulated kinases kinases (MEKK) and the phosphatidylinositol kinase [47,44,59]. IKK kinases represent a link between the “outside world” in the cell and the NFjB specific signalling pathway. IKK complex consists of two similar subunits IKKa and IKKb. IKKc, formerly known as NFjB essential modifier (NEMO) is important for some of the activation pathways of IKK. It is
Table 1 Extracellular stimuli for NFjB activation and NFjB regulated genes Extracellular stimuli TNFa Interleukin 1 ROS UV light Ischaemia Lipopolysaccharide Bacteria Viruses Amyloid Glutamate
Regulated genes Growth factors (G/M-CSF) G/M CSF, M CSF, G CSF Cell adhesion molecules ICAM-1, VCAM, E-Selectin, P-selectin Cytokines TNFa, IL-1, IL-2, IL-6, interferon Transcription regulators P53, IjB, c-rel, c-myc Antiapoptotic proteins TRAF-1, TRAF-2, c-IAP1, c-IAP2
also a target for nouvelle pharmaceutical agents. Small peptides that are cell membrane permeable and interact with NEMO, inhibit the NFjB activation [46]. IKKa and IKKb form heterodimers and though the subunits have similar structures they have different functions [15,29]. IKKb phosphorylates IjB in response to proinflammatory stimuli and stress, but also after interactions with the cell protective carbon monoxide—the product of heme oxygenase [9]. IKKa has the same activity during the development in re-
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sponse to morphogenic signals in skin and skeletal development. This seems to be evolutionary conserved as in Drosophila embryonic development IKKa plays a crucial role in the morphogenesis, which has been confirmed in gene knockout studies [28,66,76]. However, nothing is as simple as it seems. Recent findings have showed the potential of IKK and a number of other kinases (casein kinase II, mitogen and stress activated protein kinases, protein kinase C, etc.) to activate NFjB in an IjB independent manner by direct phosphorylation at various sites. The meaning of this alternative activation is not clear by now [63]. Phosphorylated IjB changes its tertiary structure and exposes hidden motifs that are recognized by ubiquitin ligase. The ubiquitin system is a conserved complex that functions as a dustman in the cell, where it marks old or defect proteins for cleavage. The evolution started to use the system as a regulation point in multicellular organisms. Thus ubiquitated IjB is marked and consecutively cleaved by the 26S proteasome [33]. After the release, NFjB is transported to the nucleus where it binds specifically to jB enhancer elements of DNA and alters the expression of a great number of genes (Table 1). Protein kinase A and p300/CBP belong to transcription coactivators that regulate the binding of NFjB in the nucleus. This is an important meeting point of NFjB and cell cycle regulation, as p300 is a substrate for cyclin dependent kinases (CDK) and it affects the RelA subunit of NFjB [55]. The termination of NFjB activation was thought to be regulated by a classic feedback pathway, while one of the jB sites is an enhancer of IjB gene. Thus, the activation of NFjB results in an enhanced expression of the inhibitor IjB and export of the NFjB–IjB complex from the nucleus to the cytoplasm. Recently, another mechanism of regulation of the intranuclear action of NFjB has been found. The cofactors p300 and CBP stimulate acetylation of NFjB, what dramatically decreases the affinity to IjB. The deacetylation necessary for the termination of the NFjB action is catalysed by histone deacetylase 3 [11]. A contradictory regulative action have small Ras-like proteins that act as guanosine triphosphatases, interact with the NFjB–IjB complex and slow down the degradation of IjB keeping NFjB in cytoplasm in an inactive state [17]. A similar effect is assumed for a totally different agent—a-tocopherol [10]. Pathogens induce NFjB activation. Particularly, viruses that are recognized by the host cells through their toll-like receptors, that are activated after the interaction with dsRNA, induce a cascade that in the end activates NFjB and enhances the expression of for example interferon a [3]. Mycoplasmas start the same immunological cascade with the lipoproteins on their outer membrane [61]. A very interesting finding is that this option is used in a similar manner in the interactions between gastrointestinal bacteria and their host. Procaryotic products inhibit the ubiquitation of IjB. The abortion of NFjB pathway has a profound anti-inflammatory effect enabling the symbiosis of procaryonts and the gastrointestinal mucosa of the host [52].
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3. Pathophysiology and clinical applications Reactive oxygen species (ROS) are toxic and in conditions of a dysbalance between their overproduction and the diminished activity of various antioxidant enzymes and other molecules induce cellular injury termed oxidative stress. ROS are often related to a number of diseases like atherosclerosis, cancer, Alzheimer’s, etc. However, the clear pathomechanism is not clear at all. Latest years of research have brought the idea of connection between ROS and NFjB. And indeed, in vitro studies showed a rapid activation of NFjB after exposure of certain cell types to ROS [14]. Today, no specific receptor for ROS has been found, thus, the details of the ROS induced activation of NFjB are missing. A hypothesis of NFjB acting as a direct intracellular receptor for ROS arises [37]. Neurodegenerative disorders are both, the result of enhanced ROS production and chronic inflammatory diseases. Thus, they are assumed to benefit from NFjB based therapy. The contemporary used drugs affect the complex IjB/NFjB non-specifically. Future specific medications are awaited [45,39]. Reperfusion injury—a process that starts a number of cell damaging actions induces also ROS production. In an experimental in vivo model of stroke, the activation of NFjB correlated with the size of the necrotic tissue. Knockout mice p50–/– on the other hand showed reduced ischaemic damage [64]. Oxygen and glucose deprivation induced apoptosis of neuronal cells is suppressed by activated NFjB [75]. Similar results were achieved in experimental seizures on neuronal tissue [2], during potassium withdrawal of cerebellar granule cells [56] and in migraine attacks [60]. In reperfusion injury of the heart a further process was investigated—the expression of adhesion molecules like ICAM-1. Evidence was brought that NFjB activation induces a rapid rise of ICAM-1 in the myocardial infarction [68]. The adaptation of cells to hypoxia is provided by the effects of hypoxia inducible factor 1. Its expression belongs to the NFjB dependent responses during the ischaemic phase of ischaemia-reperfusion injury [32]. Therapeutical agents like aspirin or heparin often successfully used in myocardial infarction act via the inhibition of NFjB [70]. Acetylsalicylic acid and salicylate salts used in chronic inflammatory diseases have been shown to inhibit NFjB activation by an unknown mechanism [23]. The results from studies about the effects of aspirin are somehow puzzling. Colorectal cancer cells undergo apoptosis mediated by NFjB activation in response to aspirin treatment [67], but NFjB activation is needed for the readhesion of metastatic cells of the same tumour type [62]. Latest in vivo studies revealed that high doses of aspirin, on the other hand, inhibit NFjB, even in cells stimulated by angiotensin II through a mechanism involving the inhibition of IKK [50]. This question of mechanism is neither answered in some other agents with similar effects. A good example is the polyphenolic compound resveratrol naturally occurring in grapes or wine. Resveratrol is often thought to be responsible for the anti-
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inflammatory, anticarcinogenic and other effects of wine and grape juices. The mechanism of action seems to be partly explained by the inhibition of TNFa induced NFjB activation, but resveratrol also inhibits apoptosis in cell in vitro, thus, resveratrol affects several cellular mechanisms [43]. Transgene mice with an altered IjB that cannot be degradated show high susceptibility to systemic infection, even though the transgene cells were restricted to hepatocytes [34]. Of high clinical relevance are the findings from studies concerning the relationship between NFjB and glucocorticoids. As immunosuppressive agents it is not surprising that glucocorticoids inhibit the activation of NFjB. However, no consensus exists yet, which pathway is responsible for this effect. These frequently indicated and used drugs can act by inducing IjB expression or by direct interaction of activated glucocorticoid receptor with NFjB inside the nucleus [16]. Further molecular link between the glucocorticoid receptor and NFjB may represent the activator protein-1 [22]. Other steroids receptors like the androgen, estrogen or progesterone receptors act probably in a similar manner [48]. Even in estrogen receptor negative breast cancer NFjB represents the target for further pharmacological interventions [8]. The research in this area is of an enormous value for future therapeutical modalities. Targeting specific down stream immune system regulation points may soon substitute the standard immunosuppressive therapy, what is important due to the broad indications, due to the unwanted side effects of steroid therapy and especially due to the number of patients taking these drugs. The complex immune regulation is unfortunately not so easy to overcome. Paper published in Nature Medicine indicates future problems of NFjB inhibitors as inflammatory agents. The resolution of inflammation is connected with apoptosis of leukocytes that is mediated by NFjB. The inhibition of this process prolongs the inflammatory process [35]. This example of proapoptotic activity of NFjB advises us to pay attention to the side effects and to hold back the enthusiasm. The outcome of polymicrobial sepsis, a severe clinical problem is also thought to be improved by NFjB inhibition, but early and late phase of sepsis must be distinguished, while the activation of the nuclear factor is phase specific as shown previously [74]. The outstanding role of NFjB as a signal transducer was confirmed in vitro. TNFa induces apoptosis in cells with inhibited transcription. In most cells, however, transcription is present under physiological circumstances. It was established that the protective protein that is both, included in the TNFa signalling pathway and antagonist of TNFa from functional point of view is NFjB. Some types of stem cells, central neurons during development, but also tumour cells from hepatocellular carcinoma were shown to have permanent active NFjB, that induces the expression of proteins of mitochondrial stability like Bcl-2 and A1, what may partly explain their resistance to apoptotic signals [6,26,69]. RelA is the mystery protein that decides about life or death of the
cell. In lymphocytes from RelA knockout mice TNFa induces rapid apoptosis. If RelA is provided back to the cells, they become resistant against TNFa induced apoptosis [5]. Similar results were found in eosinophils [19]. The TNFa vs. NFjB relationship is under investigation. It seems that a cytoplasmatic zinc finger protein called A20 may play a further link between TNFa signalling pathway and NFjB [36]. Signal transducer and transcriptional factor—Stat5b also inhibits the TNFa induced NFjB activation, which reveals the crossroad of two transcription factors—Stat and Rel [40]. But the exceptional discovery of Van Antwerp et al. [71] that cells with a dominant negative inhibitor of NFjB– IjBaM are more susceptible to TNFa induced apoptosis made clear that the NFjB signalling pathway is a natural suppressor of TNFa cytotoxicity. The same was assumed for Fas/Fas ligand induced apoptosis. However, this assumption was disproved, as Fas-mediated apoptosis is not inhibited by NFjB activation [71]. The mechanism of action of the antagonism between TNFa and NFjB was made clear later in 1998. Wang et al. [73] showed that inhibitors of apoptosis c-IAP1 and c-IAP2 as well as the antiapoptotic TNFa receptor associated factors 1 and 2 belong to proteins which expression is up regulated by activated NFjB. What is the clinical outcome of the research of the relationship between TNFa and NFjB? The use of TNFa antibodies in chronic inflammatory diseases like morbus Crohn, colitis ulcerosa or rheumatoid arthritis becomes only slowly a gold standard due to the “golden” costs [77]. Inhibiting NFjB, particularly using molecular biology, may be much cheaper and, thus, available for a wider spectrum of patients. Therapy of these “non-specific” diseases turns out to be more specific. The recent opinions regarding the role and the possible future directions of NFjB use in chronic inflammation is reviewed [41]. TNFa signalling pathway is an important player in cancerogenesis and hence, a key target for antitumour therapy. The rationale is clear. TNFa induces apoptosis, NFjB up regulates the expression of antiapoptotic molecules. The inhibition of NFjB in the tumour cells increases the sensitivity of the cells to TNFa and consequently to chemotherapy, but not to natural killer cell mediated cytotoxicity [42]. The inhibition can be achieved by different ways, one of them is the use of gene transfer by adenoviral vectors containing the artificial super-repressor form of IjB [72], another one is the use of synthetic membrane permeable protein inhibitors of NFjB [49]. The comparison of different modalities to pharmacological NFjB inhibition remains a task for the future. NFjB regulates the expression of another enzyme related to inflammation—the inducible nitric oxide synthase (iNOS), showing the width of immune system regulation by NFjB [20]. The enhanced expression of iNOS is stimulated for example by interleukin 1b. The resulting prolonged activation of NFjB is inhibited by inhibitors of MAPK and ERK1/2 indicating a role for NFjB in the development of atherosclerotic plaques and endothelial dysfunction after arterial wall injury due to inflammatory processes [30]. Car-
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diac hypertrophy induced by the stimulation of G protein coupled receptors involves the apoptosis signal regulating kinase and the activation of NFjB. This system seems to play an outstanding role in the process of atherogenesis. The future therapy of ischaemic heart disease may include inhibitors of NFjB [13,27]. Some types of inherited muscular dystrophy are related to calpain-3 deficiency. In muscle cells from the patients an alternation of the NFjB/IjB system was found. Calpain-3 seems to be partly involved in the process of sequestration of IjB, thus, calpain-3 deficient cells accumulate high amounts of IjB both, in cytoplasma and in the nucleus. The permanent inactivation of NFjB results in high frequency of programmed cell death and to muscle loss [4]. Not only inherited muscle diseases but also the muscle loss due to cachexia is related to alternation of NFjB [25]. Pneumological applications of NFjB like asthma, respiratory distress syndrome, infections and cystic fibrosis have been reviewed previously [12]. A variety of endocrinological disturbances is associated with higher levels of cytokines that are regulated by NFjB. Euthyroid sick syndrome with high levels of triiodothyronine is one of them. Clarithromycin used in this entity because of the clinical efficiency acts via inhibition of NFjB that is without therapy activated by TNFa [51]. Last but not least, diabetes mellitus seems to be related to a sustained activation of NFjB resulting in classic diabetic complications like nephropathy, neuropathy and angiopathy. These complications are very likely to be caused by the effects of toxic compounds produced by the non-enzymatic glycation, the so-called advanced glycation end products (AGEs). AGEs are a heterogeneous group of compounds that rise with the age and are markers of the carbonyl stress. Especially long-lasting high glycaemia and oxidative stress induce their production. At cellular level AGEs (whether free or bound to erythrocytes) activate the receptor for AGEs (RAGE). RAGE activates further the IKK complex by a still unknown mechanism. Under these conditions one of the genes up regulated by NFjB is endothelin 1 leading to hypertension, endothelial cell damage and atherosclerosis [58]. Therapeutical possibilities include alternation of the renin angiotensin system and the use of so-called AGE breakers like aminoguanidine [18]. Interestingly, hyaluronic acid seems to play the protective role against this pathway. But its molecular weight and so its protective activity decreases with age [53]. Though short term activation is mediated by the degradation of IjB, long term stimulation of the same signalling pathway enhance the expression of NFjB with stable initial expression and degradation rate of IjB [7]. This may be true also for other pathophysiological entities, but in vivo experiments and clinical studies are needed to clarify this assumption. Experimental and very likely clinical diabetes mellitus type I also is induced through B pancreatic islets damage. This can be protected using NFjB decoys— oligonucleotides that are copies of the NFjB DNA binding sites. These small molecules represent a nouvelle therapeti-
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cal approach in the inhibition of transcription factors [57]. This was also shown for UV light induced damage of the skin [1] and is assumed to be useful in renal diseases like the nephritic syndrome and glomerulonephritis [24,38].
4. Conclusion The complex net of signals in the cell has several checkpoints that are of greatest importance for the understanding of intracellular and intercellular regulation. From the biomedical point of view they represent the future targets of pharmacological interventions. NFjB signalling belongs to these crossroads. The functions in the immune system, mediation and regulation of inflammation responses of the cells as well as the cell cycle management makes it interesting for the whole spectrum of medical research fields. Especially, the immunosuppressive treatment that is widely used nowadays must be revised and NFjB is a suitable target for further involvement in clinical medicine. Moreover, the sensibilization of cancer cells to chemotherapy by the use of NFjB inhibitors provides a nouvelle concept for the cure of oncological diseases. Even in cardiovascular, metabolic and renal medicine, there is a place for NFjB, while it provides new insights into the pathogenesis of the most frequent disorders like atherosclerosis, diabetes mellitus and renal failure. The possibilities are nearly infinite. Old remedies used for decades are shown one by one to affect NFjB on various ways. Natural occurring agents which actions are still a matter of debate in the theory and nouvelle small molecular derivates activate or inhibit the transcriptional factor. Synthetic oligoand polypeptide inhibitors of NFjB can penetrate the cell membrane and directly act on the Rel proteins. The most sophisticated approaches towards inhibiting the activation and translocation of NFjB into the nucleus represent gene deliveries, using plasmids or adenoviruses containing genes for various super repressors—modified IjB proteins, or socalled NFjB decoys, which interact with activated NFjB and thus, inhibit the interaction between the transcription factor and nuclear DNA enhancers. The medicine of this century is a medicine of molecules, the diagnostic procedure and the therapy moves further from the “clinical picture” to the use of achievements in molecular biology and genetics. However, sober scepticism and awareness are indicated. Especially the role of NFjB in multiple signal transducing pathways and the tissue dependent variability of responses to alternations in NFjB pathway may be the reasons for unwanted side effects of the therapy that are after in vitro or in vivo experiments hardly to expect in the clinical use.
Acknowledgements I thank Dr. Ingrid Jurkovicˇová and Mr. Július Hodosy for helpful comments and for reading and revising the manu-
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script. The author is supported by grant no. 116/2004 from Comenius University.
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Review
Nuclear factor kappa B—molecular biomedicine: the next generation Peter Celec a,b,* a
b
Institute of Pathophysiology, Faculty of Medicine, Comenius University, Bratislava, Slovakia Departmant of Molecular Biology, Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia Received 20 November 2003; accepted 24 December 2003 Available online 08 June 2004
Abstract Nuclear factor kappa B (NFjB) as a transcription factor plays an important integrating role in the intracellular regulation of immune response, inflammation and cell cycle regulation. Nouvelle insights into the structure and regulation of activation of NFjB have brought a detailed picture of the function of this transcription factor. In this review the findings of interactions of NFjB with its inhibitors, tumour necrosis factor alpha and glucocorticoids are presented. The results from the latest in vivo studies show the capability of specific NFjB inhibitors in the clinical use. This article summarizes the most important facts regarding NFjB participation in the pathogenesis of diseases and its potential as a target of pharmacological agents. © 2004 Elsevier SAS. All rights reserved. Keywords: Nuclear factor kappa B; Apoptosis; Cell cycle regulation; Immunosuppressive therapy; Inflammation
1. Introduction
2. The molecules and their regulation
In 1986 Sen and Baltimore [65] first described a nuclear protein that should capture the interest of researchers for the next decades. However, the opinion that this protein is a kappa immunoglobulin enhancer activator specific for mature B cells has changed dramatically. The functional territory has widened extraordinarily. The term nuclear factor kappa B (NFjB) remained untouched. Nowadays, it is clear that NFjB plays a central role in the regulation of cellular processes throughout the tissues. Although the function in immune competent cells is best described, the master position in apoptosis and cell cycle control explains the growing number of publications in molecular biology and biomedical research (Fig. 1). The complex of interactions with other relevant signalling pathways at cellular level and the relation to a number of pathological entities outline the hope that is put to NFjB as a potential pharmaceutical target of the next generation [54].
NFjB is not a single and unique molecule. It is more a functional description for a complex of two variable subunits that come from the Rel/NFjB family. The members (RelA, RelB, c-Rel, p100/p52 and p105/p50) contain a so-called Rel homology domain—a 300 amino acid long sequence that is responsible for the binding to the other subunit and to the
* Corresponding author: Galbaveho 3, 841 01 Bratislava, Slovakia. E-mail address: [email protected] (P. Celec). © 2004 Elsevier SAS. All rights reserved. doi:10.1016/j.biopha.2003.12.015
Fig. 1. Number of publications concerning NFjB per year as the result of a Medline search.
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Fig. 2. A simplified scheme of the activation of NFjB by the degradation of IjB. IjB is phosphorylated by IKK and ubiquinatated by the ubiquitine ligase system (ULS). IjB is further degradated by the 26S proteasome (26S). Activated NFjB can pass the nuclear membrane and interact with jB binding sequences in enhancers of NFjB regulated genes. LPS, lipopolysaccharide; ROS, reactive oxygen species; FasL, Fas ligand; TRAF, TNFa receptor associated factor; NIK, NFjB inducing kinase; MEKK, mitogen activated protein kinase/extracellular signal regulated kinases kinases.
DNA [21]. The best characterized variant is the combination of RelA and p50. Inactivated subunits are localized as homoor hetero-dimers in cytoplasm together with IjB—the NFjB inhibitor. The IjB family has four known members (IjBa, IjBb, IjBe and Bcl3). These proteins contain an ankyrin repeat motif, which is important for the maintenance of NFjB in the cytoplasm [31]. The activation of NFjB lies in the release from IjB (Fig. 2). A great number of stimuli, which are partly mentioned in Table 1, activate IjB kinase (IKK) kinases, which include NFjB inducing kinase (NIK), NFjB activating kinase (NAK), atypical protein kinases C, Akt/protein kinase B, proteins related to IKK, receptor activator for NFjB and its ligand (RANK and RANKL), but also mitogen activated protein kinase kinases (MAPKK), mitogen activated protein kinase/extracellular signal regulated kinases kinases (MEKK) and the phosphatidylinositol kinase [47,44,59]. IKK kinases represent a link between the “outside world” in the cell and the NFjB specific signalling pathway. IKK complex consists of two similar subunits IKKa and IKKb. IKKc, formerly known as NFjB essential modifier (NEMO) is important for some of the activation pathways of IKK. It is
Table 1 Extracellular stimuli for NFjB activation and NFjB regulated genes Extracellular stimuli TNFa Interleukin 1 ROS UV light Ischaemia Lipopolysaccharide Bacteria Viruses Amyloid Glutamate
Regulated genes Growth factors (G/M-CSF) G/M CSF, M CSF, G CSF Cell adhesion molecules ICAM-1, VCAM, E-Selectin, P-selectin Cytokines TNFa, IL-1, IL-2, IL-6, interferon Transcription regulators P53, IjB, c-rel, c-myc Antiapoptotic proteins TRAF-1, TRAF-2, c-IAP1, c-IAP2
also a target for nouvelle pharmaceutical agents. Small peptides that are cell membrane permeable and interact with NEMO, inhibit the NFjB activation [46]. IKKa and IKKb form heterodimers and though the subunits have similar structures they have different functions [15,29]. IKKb phosphorylates IjB in response to proinflammatory stimuli and stress, but also after interactions with the cell protective carbon monoxide—the product of heme oxygenase [9]. IKKa has the same activity during the development in re-
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sponse to morphogenic signals in skin and skeletal development. This seems to be evolutionary conserved as in Drosophila embryonic development IKKa plays a crucial role in the morphogenesis, which has been confirmed in gene knockout studies [28,66,76]. However, nothing is as simple as it seems. Recent findings have showed the potential of IKK and a number of other kinases (casein kinase II, mitogen and stress activated protein kinases, protein kinase C, etc.) to activate NFjB in an IjB independent manner by direct phosphorylation at various sites. The meaning of this alternative activation is not clear by now [63]. Phosphorylated IjB changes its tertiary structure and exposes hidden motifs that are recognized by ubiquitin ligase. The ubiquitin system is a conserved complex that functions as a dustman in the cell, where it marks old or defect proteins for cleavage. The evolution started to use the system as a regulation point in multicellular organisms. Thus ubiquitated IjB is marked and consecutively cleaved by the 26S proteasome [33]. After the release, NFjB is transported to the nucleus where it binds specifically to jB enhancer elements of DNA and alters the expression of a great number of genes (Table 1). Protein kinase A and p300/CBP belong to transcription coactivators that regulate the binding of NFjB in the nucleus. This is an important meeting point of NFjB and cell cycle regulation, as p300 is a substrate for cyclin dependent kinases (CDK) and it affects the RelA subunit of NFjB [55]. The termination of NFjB activation was thought to be regulated by a classic feedback pathway, while one of the jB sites is an enhancer of IjB gene. Thus, the activation of NFjB results in an enhanced expression of the inhibitor IjB and export of the NFjB–IjB complex from the nucleus to the cytoplasm. Recently, another mechanism of regulation of the intranuclear action of NFjB has been found. The cofactors p300 and CBP stimulate acetylation of NFjB, what dramatically decreases the affinity to IjB. The deacetylation necessary for the termination of the NFjB action is catalysed by histone deacetylase 3 [11]. A contradictory regulative action have small Ras-like proteins that act as guanosine triphosphatases, interact with the NFjB–IjB complex and slow down the degradation of IjB keeping NFjB in cytoplasm in an inactive state [17]. A similar effect is assumed for a totally different agent—a-tocopherol [10]. Pathogens induce NFjB activation. Particularly, viruses that are recognized by the host cells through their toll-like receptors, that are activated after the interaction with dsRNA, induce a cascade that in the end activates NFjB and enhances the expression of for example interferon a [3]. Mycoplasmas start the same immunological cascade with the lipoproteins on their outer membrane [61]. A very interesting finding is that this option is used in a similar manner in the interactions between gastrointestinal bacteria and their host. Procaryotic products inhibit the ubiquitation of IjB. The abortion of NFjB pathway has a profound anti-inflammatory effect enabling the symbiosis of procaryonts and the gastrointestinal mucosa of the host [52].
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3. Pathophysiology and clinical applications Reactive oxygen species (ROS) are toxic and in conditions of a dysbalance between their overproduction and the diminished activity of various antioxidant enzymes and other molecules induce cellular injury termed oxidative stress. ROS are often related to a number of diseases like atherosclerosis, cancer, Alzheimer’s, etc. However, the clear pathomechanism is not clear at all. Latest years of research have brought the idea of connection between ROS and NFjB. And indeed, in vitro studies showed a rapid activation of NFjB after exposure of certain cell types to ROS [14]. Today, no specific receptor for ROS has been found, thus, the details of the ROS induced activation of NFjB are missing. A hypothesis of NFjB acting as a direct intracellular receptor for ROS arises [37]. Neurodegenerative disorders are both, the result of enhanced ROS production and chronic inflammatory diseases. Thus, they are assumed to benefit from NFjB based therapy. The contemporary used drugs affect the complex IjB/NFjB non-specifically. Future specific medications are awaited [45,39]. Reperfusion injury—a process that starts a number of cell damaging actions induces also ROS production. In an experimental in vivo model of stroke, the activation of NFjB correlated with the size of the necrotic tissue. Knockout mice p50–/– on the other hand showed reduced ischaemic damage [64]. Oxygen and glucose deprivation induced apoptosis of neuronal cells is suppressed by activated NFjB [75]. Similar results were achieved in experimental seizures on neuronal tissue [2], during potassium withdrawal of cerebellar granule cells [56] and in migraine attacks [60]. In reperfusion injury of the heart a further process was investigated—the expression of adhesion molecules like ICAM-1. Evidence was brought that NFjB activation induces a rapid rise of ICAM-1 in the myocardial infarction [68]. The adaptation of cells to hypoxia is provided by the effects of hypoxia inducible factor 1. Its expression belongs to the NFjB dependent responses during the ischaemic phase of ischaemia-reperfusion injury [32]. Therapeutical agents like aspirin or heparin often successfully used in myocardial infarction act via the inhibition of NFjB [70]. Acetylsalicylic acid and salicylate salts used in chronic inflammatory diseases have been shown to inhibit NFjB activation by an unknown mechanism [23]. The results from studies about the effects of aspirin are somehow puzzling. Colorectal cancer cells undergo apoptosis mediated by NFjB activation in response to aspirin treatment [67], but NFjB activation is needed for the readhesion of metastatic cells of the same tumour type [62]. Latest in vivo studies revealed that high doses of aspirin, on the other hand, inhibit NFjB, even in cells stimulated by angiotensin II through a mechanism involving the inhibition of IKK [50]. This question of mechanism is neither answered in some other agents with similar effects. A good example is the polyphenolic compound resveratrol naturally occurring in grapes or wine. Resveratrol is often thought to be responsible for the anti-
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inflammatory, anticarcinogenic and other effects of wine and grape juices. The mechanism of action seems to be partly explained by the inhibition of TNFa induced NFjB activation, but resveratrol also inhibits apoptosis in cell in vitro, thus, resveratrol affects several cellular mechanisms [43]. Transgene mice with an altered IjB that cannot be degradated show high susceptibility to systemic infection, even though the transgene cells were restricted to hepatocytes [34]. Of high clinical relevance are the findings from studies concerning the relationship between NFjB and glucocorticoids. As immunosuppressive agents it is not surprising that glucocorticoids inhibit the activation of NFjB. However, no consensus exists yet, which pathway is responsible for this effect. These frequently indicated and used drugs can act by inducing IjB expression or by direct interaction of activated glucocorticoid receptor with NFjB inside the nucleus [16]. Further molecular link between the glucocorticoid receptor and NFjB may represent the activator protein-1 [22]. Other steroids receptors like the androgen, estrogen or progesterone receptors act probably in a similar manner [48]. Even in estrogen receptor negative breast cancer NFjB represents the target for further pharmacological interventions [8]. The research in this area is of an enormous value for future therapeutical modalities. Targeting specific down stream immune system regulation points may soon substitute the standard immunosuppressive therapy, what is important due to the broad indications, due to the unwanted side effects of steroid therapy and especially due to the number of patients taking these drugs. The complex immune regulation is unfortunately not so easy to overcome. Paper published in Nature Medicine indicates future problems of NFjB inhibitors as inflammatory agents. The resolution of inflammation is connected with apoptosis of leukocytes that is mediated by NFjB. The inhibition of this process prolongs the inflammatory process [35]. This example of proapoptotic activity of NFjB advises us to pay attention to the side effects and to hold back the enthusiasm. The outcome of polymicrobial sepsis, a severe clinical problem is also thought to be improved by NFjB inhibition, but early and late phase of sepsis must be distinguished, while the activation of the nuclear factor is phase specific as shown previously [74]. The outstanding role of NFjB as a signal transducer was confirmed in vitro. TNFa induces apoptosis in cells with inhibited transcription. In most cells, however, transcription is present under physiological circumstances. It was established that the protective protein that is both, included in the TNFa signalling pathway and antagonist of TNFa from functional point of view is NFjB. Some types of stem cells, central neurons during development, but also tumour cells from hepatocellular carcinoma were shown to have permanent active NFjB, that induces the expression of proteins of mitochondrial stability like Bcl-2 and A1, what may partly explain their resistance to apoptotic signals [6,26,69]. RelA is the mystery protein that decides about life or death of the
cell. In lymphocytes from RelA knockout mice TNFa induces rapid apoptosis. If RelA is provided back to the cells, they become resistant against TNFa induced apoptosis [5]. Similar results were found in eosinophils [19]. The TNFa vs. NFjB relationship is under investigation. It seems that a cytoplasmatic zinc finger protein called A20 may play a further link between TNFa signalling pathway and NFjB [36]. Signal transducer and transcriptional factor—Stat5b also inhibits the TNFa induced NFjB activation, which reveals the crossroad of two transcription factors—Stat and Rel [40]. But the exceptional discovery of Van Antwerp et al. [71] that cells with a dominant negative inhibitor of NFjB– IjBaM are more susceptible to TNFa induced apoptosis made clear that the NFjB signalling pathway is a natural suppressor of TNFa cytotoxicity. The same was assumed for Fas/Fas ligand induced apoptosis. However, this assumption was disproved, as Fas-mediated apoptosis is not inhibited by NFjB activation [71]. The mechanism of action of the antagonism between TNFa and NFjB was made clear later in 1998. Wang et al. [73] showed that inhibitors of apoptosis c-IAP1 and c-IAP2 as well as the antiapoptotic TNFa receptor associated factors 1 and 2 belong to proteins which expression is up regulated by activated NFjB. What is the clinical outcome of the research of the relationship between TNFa and NFjB? The use of TNFa antibodies in chronic inflammatory diseases like morbus Crohn, colitis ulcerosa or rheumatoid arthritis becomes only slowly a gold standard due to the “golden” costs [77]. Inhibiting NFjB, particularly using molecular biology, may be much cheaper and, thus, available for a wider spectrum of patients. Therapy of these “non-specific” diseases turns out to be more specific. The recent opinions regarding the role and the possible future directions of NFjB use in chronic inflammation is reviewed [41]. TNFa signalling pathway is an important player in cancerogenesis and hence, a key target for antitumour therapy. The rationale is clear. TNFa induces apoptosis, NFjB up regulates the expression of antiapoptotic molecules. The inhibition of NFjB in the tumour cells increases the sensitivity of the cells to TNFa and consequently to chemotherapy, but not to natural killer cell mediated cytotoxicity [42]. The inhibition can be achieved by different ways, one of them is the use of gene transfer by adenoviral vectors containing the artificial super-repressor form of IjB [72], another one is the use of synthetic membrane permeable protein inhibitors of NFjB [49]. The comparison of different modalities to pharmacological NFjB inhibition remains a task for the future. NFjB regulates the expression of another enzyme related to inflammation—the inducible nitric oxide synthase (iNOS), showing the width of immune system regulation by NFjB [20]. The enhanced expression of iNOS is stimulated for example by interleukin 1b. The resulting prolonged activation of NFjB is inhibited by inhibitors of MAPK and ERK1/2 indicating a role for NFjB in the development of atherosclerotic plaques and endothelial dysfunction after arterial wall injury due to inflammatory processes [30]. Car-
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diac hypertrophy induced by the stimulation of G protein coupled receptors involves the apoptosis signal regulating kinase and the activation of NFjB. This system seems to play an outstanding role in the process of atherogenesis. The future therapy of ischaemic heart disease may include inhibitors of NFjB [13,27]. Some types of inherited muscular dystrophy are related to calpain-3 deficiency. In muscle cells from the patients an alternation of the NFjB/IjB system was found. Calpain-3 seems to be partly involved in the process of sequestration of IjB, thus, calpain-3 deficient cells accumulate high amounts of IjB both, in cytoplasma and in the nucleus. The permanent inactivation of NFjB results in high frequency of programmed cell death and to muscle loss [4]. Not only inherited muscle diseases but also the muscle loss due to cachexia is related to alternation of NFjB [25]. Pneumological applications of NFjB like asthma, respiratory distress syndrome, infections and cystic fibrosis have been reviewed previously [12]. A variety of endocrinological disturbances is associated with higher levels of cytokines that are regulated by NFjB. Euthyroid sick syndrome with high levels of triiodothyronine is one of them. Clarithromycin used in this entity because of the clinical efficiency acts via inhibition of NFjB that is without therapy activated by TNFa [51]. Last but not least, diabetes mellitus seems to be related to a sustained activation of NFjB resulting in classic diabetic complications like nephropathy, neuropathy and angiopathy. These complications are very likely to be caused by the effects of toxic compounds produced by the non-enzymatic glycation, the so-called advanced glycation end products (AGEs). AGEs are a heterogeneous group of compounds that rise with the age and are markers of the carbonyl stress. Especially long-lasting high glycaemia and oxidative stress induce their production. At cellular level AGEs (whether free or bound to erythrocytes) activate the receptor for AGEs (RAGE). RAGE activates further the IKK complex by a still unknown mechanism. Under these conditions one of the genes up regulated by NFjB is endothelin 1 leading to hypertension, endothelial cell damage and atherosclerosis [58]. Therapeutical possibilities include alternation of the renin angiotensin system and the use of so-called AGE breakers like aminoguanidine [18]. Interestingly, hyaluronic acid seems to play the protective role against this pathway. But its molecular weight and so its protective activity decreases with age [53]. Though short term activation is mediated by the degradation of IjB, long term stimulation of the same signalling pathway enhance the expression of NFjB with stable initial expression and degradation rate of IjB [7]. This may be true also for other pathophysiological entities, but in vivo experiments and clinical studies are needed to clarify this assumption. Experimental and very likely clinical diabetes mellitus type I also is induced through B pancreatic islets damage. This can be protected using NFjB decoys— oligonucleotides that are copies of the NFjB DNA binding sites. These small molecules represent a nouvelle therapeti-
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cal approach in the inhibition of transcription factors [57]. This was also shown for UV light induced damage of the skin [1] and is assumed to be useful in renal diseases like the nephritic syndrome and glomerulonephritis [24,38].
4. Conclusion The complex net of signals in the cell has several checkpoints that are of greatest importance for the understanding of intracellular and intercellular regulation. From the biomedical point of view they represent the future targets of pharmacological interventions. NFjB signalling belongs to these crossroads. The functions in the immune system, mediation and regulation of inflammation responses of the cells as well as the cell cycle management makes it interesting for the whole spectrum of medical research fields. Especially, the immunosuppressive treatment that is widely used nowadays must be revised and NFjB is a suitable target for further involvement in clinical medicine. Moreover, the sensibilization of cancer cells to chemotherapy by the use of NFjB inhibitors provides a nouvelle concept for the cure of oncological diseases. Even in cardiovascular, metabolic and renal medicine, there is a place for NFjB, while it provides new insights into the pathogenesis of the most frequent disorders like atherosclerosis, diabetes mellitus and renal failure. The possibilities are nearly infinite. Old remedies used for decades are shown one by one to affect NFjB on various ways. Natural occurring agents which actions are still a matter of debate in the theory and nouvelle small molecular derivates activate or inhibit the transcriptional factor. Synthetic oligoand polypeptide inhibitors of NFjB can penetrate the cell membrane and directly act on the Rel proteins. The most sophisticated approaches towards inhibiting the activation and translocation of NFjB into the nucleus represent gene deliveries, using plasmids or adenoviruses containing genes for various super repressors—modified IjB proteins, or socalled NFjB decoys, which interact with activated NFjB and thus, inhibit the interaction between the transcription factor and nuclear DNA enhancers. The medicine of this century is a medicine of molecules, the diagnostic procedure and the therapy moves further from the “clinical picture” to the use of achievements in molecular biology and genetics. However, sober scepticism and awareness are indicated. Especially the role of NFjB in multiple signal transducing pathways and the tissue dependent variability of responses to alternations in NFjB pathway may be the reasons for unwanted side effects of the therapy that are after in vitro or in vivo experiments hardly to expect in the clinical use.
Acknowledgements I thank Dr. Ingrid Jurkovicˇová and Mr. Július Hodosy for helpful comments and for reading and revising the manu-
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script. The author is supported by grant no. 116/2004 from Comenius University.
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