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Review
. 2014 Feb 28:2:832-46.
doi: 10.1016/j.redox.2014.02.008. eCollection 2014.

Concerted action of Nrf2-ARE pathway, MRN complex, HMGB1 and inflammatory cytokines - implication in modification of radiation damage

Anuranjani  1 , Madhu Bala  1
Affiliations
Review

Concerted action of Nrf2-ARE pathway, MRN complex, HMGB1 and inflammatory cytokines - implication in modification of radiation damage

Anuranjani et al. Redox Biol. .

Abstract

Whole body exposure to low linear energy transfer (LET) ionizing radiations (IRs) damages vital intracellular bio-molecules leading to multiple cellular and tissue injuries as well as pathophysiologies such as inflammation, immunosuppression etc. Nearly 70% of damage is caused indirectly by radiolysis of intracellular water leading to formation of reactive oxygen species (ROS) and free radicals and producing a state of oxidative stress. The damage is also caused by direct ionization of biomolecules. The type of radiation injuries is dependent on the absorbed radiation dose. Sub-lethal IR dose produces more of DNA base damages, whereas higher doses produce more DNA single strand break (SSBs), and double strand breaks (DSBs). The Nrf2-ARE pathway is an important oxidative stress regulating pathway. The DNA DSBs repair regulated by MRN complex, immunomodulation and inflammation regulated by HMGB1 and various types of cytokines are some of the key pathways which interact with each other in a complex manner and modify the radiation response. Because the majority of radiation damage is via oxidative stress, it is essential to gain in depth understanding of the mechanisms of Nrf2-ARE pathway and understand its interactions with MRN complex, HMGB1 and cytokines to increase our understanding on the radiation responses. Such information is of tremendous help in development of medical radiation countermeasures, radioprotective drugs and therapeutics. Till date no approved and safe countermeasure is available for human use. This study reviews the Nrf2-ARE pathway and its crosstalk with MRN-complex, HMGB1 and cytokines (TNF-a, IL-6, IFN-? etc.). An attempt is also made to review the modification of some of these pathways in presence of selected antioxidant radioprotective compounds or herbal extracts.

Keywords: .OH, hydroxyl radical; AP1, activator protein-1; ARE, antioxidant response element; ATM, ataxia telangiectasia mutagenesis; Bcl-2, B cell lymphoma-2 protein; CBP, CREB-binding protein; Chk-2, checkpoint kinase-2 protein; DAMP, death associated molecular pattern; DDR, DNA damage response; DGR, double glycine repeats; DSB, double strands break; FGF, fibroblast growth factor; FGF2, fibroblast growth factor-2; GM-CSF, granulocytes macrophages colony stimulating factor; GPx, glutathione peroxidase; GSH, glutathione (reduced); GSK-3ß, glycogen synthase kinase 3 beta; HMGB1; HMGB1, high mobility group Box 1; HR, homologous recombination; IR, ionizing radiation; Keap1, Kelch like ECH associated protein 1; LET, linear energy transfer; MDA, malondialdehyde; MIP, macrophages inflammatory proteins; MRN complex; MRN, Mre11, Rad50 and Nbs1 subunits; MRP, multidrug resistance protein; NADPH, nicotinamide adenine dinucleotide phosphate; NES, nuclear export sequence; NHEJ, non-homologous end joining; NLS, nuclear localization sequence; Nrf2-ARE pathway; PKC, protein kinase C; RAGE, receptor for advance glycation end products; RIF, radiation induced foci; RNS, reactive nitrogen species; ROS, reactive oxygen species; Radio-modification; SOD, superoxide dismutase; SSBs, single strand DNA breaks; TRAIL, TNF related apoptosis inducing ligand; TWEAK; TWEAK, tumour necrosis factor weak inducer of apoptosis; VEGF, vascular endothelial growth factor; VSMC, vascular smooth muscle cells; bFGF, basal fibroblast growth factor; t-BHQ, tert butyl hydroquinone.

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Figures

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Graphical abstract
Fig. 1
Fig. 1
Schematic presentation of ROS mediated lipid peroxidation (LPx) chain reaction. The figure also shows the formation of highly reactive lipid peroxyl radicals (LPO.) such as malondialdehyde (MDA) and 4-hydroxy-2(E)-nonenal (4HNE); as well as the influence of anti-oxidants on the LPx chain reactions. Ascorbic acid (vitamin C) and alpha-tocopherol (vitamin E) neutralize LPO. and act as antioxidants. Vitamin C serves dual role of pro-oxidant and antioxidant. As a pro-oxidant, the vitamin C catalyses conversion of lipid hydroperoxides (LOOH) into toxic LPO.. The toxic LPO. can damage the macromolecules (DNA, RNA, proteins) and may initiate cytotoxic, genotoxic and inflammatory reactions. The anti-oxidant property of vitamin C helps to prevent the interaction of lipid peroxidation products with the macromolecules (DNA, RNA, proteins) by converting them into unreactive conjugate of vitamin C-LPO products. Vitamin C also helps in the regeneration of a-tocopherol (vitamin E) through redox reaction. Vitamin C donates electron to tocopheryl radical (vitamin-E–O.) and therefore, helps in maintaining the intracellular concentration of reduced tocopherol.
Fig. 2(A)
Fig. 2(A)
Schematic presentation of Nrf2 structure, which consists six highly conserved regions called neh (Nrf2-ECH homology) domains. The NMR structural analysis demonstrated that Neh2 region of Nrf2 is flanked by low affinity DLG motif and high affinity ETGE motif in a-helix conformation. The DLG motif is conserved among members of the CNC-bZip family members. The ETGE motif of Neh2 possess strong binding affinity towards Keap1–Kelch domain [37]. Katoh et al. [38] demonstrated that Neh4 and Neh5 domains are two independent domains, which are rich in acidic residue and interact with CREB-binding protein (CBP). The Neh6 domain is indispensable. The Neh1 domain of Nrf2 allows for binding and heterodimerization with small Maf proteins within the nucleus. This modified complex of Nrf2 binds on upstream region of ARE sequence of DNA, under oxidative/electrophilic stress conditions. The C-terminal Neh3 is indispensable for transcriptional activity. To initiate the transcriptional activity Neh3 recruits chromo domain helicase  DNA binding protein-6 (CHD6) [39], which is a chromatin remodelling enzyme and a DNA dependent ATPase associated with RNA polymerase II during transcription initiation and elongation.
Fig. 2(B)
Fig. 2(B)
Diagrammatic presentation to explain the domains and their sequence pattern in Keap1 dimer structure. The structural conformation of Keap1 exists in the form of ß-sheets. (b) At N-terminal BTB/POZ domain of Keap1 binds to another homomer from other N-terminal BTB/POZ domain, stabilizes the dimer structure and modifies the Kelch domains to hold Nrf2 at C-terminal, so that Cul3–Rbx–E3 ligase component degrades the Nrf2 under normal/unstressed conditions. Under normal redox conditions, BTB/POZ domain of Keap1 performs two functions mainly i.e. (i) formation of Keap1 homodimers (ii) provide binding site to Cul3–E3 ubiquitin ligase which regulates Nrf2 degradation. C151 residue (located at Cul3–E3 ligase binding site on BTB/POZ) is necessary to provide active binding site to the ubiquitin complex. (c) C273 and C288 residues present on IVR region are essential to maintain E3-ligase ubiquitin activity. (d) The C-terminal Kelch repeats physically attaches with Neh2 domain of Nrf2 [43].
Fig. 3
Fig. 3
Diagrammatic presentation of regulation of Keap1-Nrf2-ARE pathway. Under the oxidative stress condition, disruption of the Keap1 dimer occurs due to the mutation on ser-104 residue of BTB/POZ domain of Keap1 [59]. In turn protein kinase C phosphorylates Nrf2–Neh2 domain at ser-40 residue, which is essentially required for complete release of Nrf2 from Keap1 dimer into the cytoplasm [52]. The free form of Nrf2 showed disabled NES motifs and activation of NLS. The NESTA, which was disabled due to the sulphhydryl modification on C183 residue, caused steric hindrance upon interaction with Crm1/exportin [60]. The NESZIP motif, however, turned off due to the masking of export sequences when Nrf2 heterodimerize with small Maf proteins within the nucleus [61]. NLS motif on Nrf2 is recognized by the importin a5 and ß1 receptor homology on nuclear membrane for entering into the nucleus [57,61]. Within the nucleus, core sequence (5'-TGACnnm GC-3’) of cis-acting regulatory element ARE binds to the Nrf2 (5'-TGA(C/G)TCA-3') with a complementary sequence homology. The Nrf2–ARE regulates transcription of antioxidants and phase II detoxifying enzymes which in turn play important role in cellular defence. Upon achieving redox homeostasis, the Keap1 independently enters into the nucleus via the receptor importin alpha-7 (also known as KPNA6) recognized by the C-terminal Kelch repeats having NLS motifs. The nuclear Keap1 mediates Nrf2 dissociation from ARE. Subsequently, Nrf2 NES activation directs the exporting of Keap1–Nrf2 complex out of the nucleus via Crm1/exportin [62]. The Keap1–Nrf2 complex bind to the Cul3–E3 ligase ubiquitin core complex within the cytosol resulting in further ubiquitination and degradation of Nrf2.
Fig. 4
Fig. 4
A mechanism showing tyrosine kinase receptor induced regulation of Nrf2 nuclear–cytoplasmic shuttling under redox homeostasis conditions. The cytoplasmic signal transduction (blue) of GSK3ß regulates the Nrf2 phosphorylation and its transfer from nucleus (pink) to cytoplasm. The GSK3ß acts as an upstream regulator for Src-family kinases which include Fyn/Src/Lyn/Fgr kinases. Gradually, when cell achieves the redox homeostasis state, the activated GSK-3ß phosphorylates Fyn kinase at threonine residue to promote nuclear localization of Fyn. The phosphorylated Fyn kinase further phosphorylates Nrf2 Try568 residue within the nucleus, which result in Nrf2 separation from ARE leading to export/degradation of Nrf2 from the nucleus. On the other hand, during early response of oxidative stress, PKC mediated phosphorylation of Nrf2 at ser-40, inactivated the GSK3ß due to the phosphorylation at ser-9 residue and only basal level of Fyn kinase expression was detected. The Fyn kinases were auto-phosphorylated at Tyr213 residue via unknown tyrosine kinase signalling. The Tyr-213 phosphorylated Fyn exported from nucleus via Crm1-exportin so that Nrf2–ARE expression was enhanced in early response to oxidative stress conditions [65] Fyn acted as an inhibitor of Nrf2–ARE complex at a later stage of oxidative stress when cell is trying to attain a redox homeostasis (delayed response).
Fig. 5
Fig. 5
Schematic presentation of MRN complex recruitment towards radiation induced DNA damage foci. The MRN complex initiates a cascade of phosphorylation events, which activate DNA damage response (DDR) protein kinases for cell cycle arrest during repair. Ionizing radiation induced foci (RIF) on DNA generated due to the phosphorylation of conserved histone H2A variant, H2AX at serine 139 residue [86]. The phosphorylated form ?-H2AX covered the large region of chromatin including the damaged site per DNA within the few minutes and reached at peak in 30 min of IR exposure [87]. The C-terminal of NBS1 101 amino acid sequences are the strongly interacting sites for Mre11 as well as are the sites for attachment of ataxia telangiectasia mutant (ATM), an another DNA repair protein [88]. The MRN mediated DNA end processing also generates small ssDNA oligomers due to the endonuclease activity of Mre11 subunit [89]. Under normal conditions, ATM dimer composition (inactive form) exits within the cytoplasm whereas DDR initiate autophosphorylation at ser-1981 residue of ATM. In reverse, ATM phosphorylated the MR bound NBS1 subunit at ser-343 residue. The phosphorylation events form an active MRN–ATM complex. The MRN–ATM complex binds to the RIF to stimulate the downstream DDR pathway. At the same time, several other protein kinases also are recruited towards RIF i.e. mediator of DNA damage CHK1 (MDC1), binding protein1 (53BP1) which directly link up with the ?H2AX foci. The phosphorylated ATM is attached with FHA domain of MDC1 protein that primarily holds the ?-H2AX with its other domain BRCT. The activated ATM phosphorylated the cell cycle checkpoint 2 (Chk2) at Ther-68 residue. The phosphorylated Chk2 mediated cdc25C phosphorylation at ser-216 residue inhibited the cdk1 activity and thus arrested the cell cycle at the G2/M phase. ATM-Chk2 association also phosphorylate ser-20 residue of p53, thereby activating the apoptosis of severely injured unrepaired DNA. ATM mediated direct phosphorylation of p53–p21 complex at multiple sites helps in DNA repair and cell cycle arrest at the G1 phase.
Fig. 6(A)
Fig. 6(A)
Detailed structure presentation of chromatin bounded nuclear HMGB1 molecule. Under normal conditions, chromatin bound HMGB-1 nuclear export and its pro-inflammatory activity is repressed by histone deacetylate enzyme (HDAC-1) in the nucleus. The increased oxidative stress conditions leads to extracellular release of reduced form of acetylated HMGB1 by necrotic cell death, which acts as inflammatory cytokine.
Fig. 6(B)
Fig. 6(B)
Schematic presentation showing the HMGB1 release from the nucleus due to the necrotic cell death under oxidative stress conditions. The HMGB1 secretion enhanced the inflammation and initiated a cascade of signal transduction for the release of proinflammatory cytokines. The secreted extracellular HMGB1 molecule structurally consists thiols cysteine residues in its DNA binding A and B domains. The reduced form of HMGB1 acts as chemoattractant for leukocytes recruitment at the inflamed site. Under oxidative stress conditions, extracellular HMGB1 is readily oxidized and primarily forms disulphide linkage between adjacent cysteine molecules 23 and 45 on A-box. The disulphide form of HMGB1 actively stimulates the proinflammatory cytokine production via binding to RAGE receptor [114,116]. The HMGB1 is itself considered as DAMP molecule because it forms highly inflammatory complex in association with ssDNA, necrotic cell debris, IL-1ß etc. The secreted HMGB1 directly act onto the activated infiltrated immune cells which reach immediately at the necrotic site. The primary infiltered immune cells i.e. neutrophils, macrophages, monocytes possess cell surface receptor for advanced glycation end products (RAGE) specific for HMGB1 binding [117]. RAGE is a transmembrane receptor protein mainly present on immunoglobulin superfamily. RAGE–HMGB1 complex contributes to the induction of proinflammatory genes and lethal shock signalling [119]. The complex triggers the activation of NF-kB, ERK, PI3/AKT signalling and stimulates the gene expression of various IL-6, TNF-a, macrophages inflammatory proteins (MIP) 1a and 1ß proteins etc. [118,119].Cumulative effect of continuous rising oxidative stress as well as of tissue damage leads to the irreversible complete oxidation of all cys residues of HMGB1 A and B boxes. The sulphonic (oxidized) form of HMGB1 modifies its functional activity i.e. abrogates pro-inflammatory cytokine production and chemoattractant property. The oxidized HMGB1 promotes the tissue regeneration and survival response. It was demonstrated invivo and in vitro that complete oxidation of HMGB1 acted as a feedback mechanism to counter the inflammatory activity and tissue damage [114,115].
Fig. 7
Fig. 7
Proposed crosstalk between the Nrf2-ARE pathway, HMGB1 and DNA damage response (DDR). The increase in oxidative stress causes multiple effects such as inflammation, DNA damage, and immunosuppression. The Nrf2-ARE pathway regulates time kinetics of HMGB1 level. The modification of oxidative stress by Nrf2-ARE also influences DDR. The MRN complex activated as a part of DDR may further augment p53. Under oxidative stress conditions the p21 interacts with Neh2 domain of Nrf2. During DDR, MRN complex activates p53, which in turn interacts with p21. The direct evidence of p21 mediated interaction of Nrf2 and MRN is lacking. The interaction of Nrf2 with p53-Chk2 is also not clear from the available literature.
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