Macrophage polarization- a response to the microenvironment

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The Thioredoxin System

In 1964, Trx1 was first isolated from Escherichia coli by Laurent et al reporting that E. coli also contained an enzyme, TrxR, which catalyzes its reduction 56. Through its redox-active cysteine residues, the dithiol Trx plays a vital role in maintaining the cellular environment in a reduced state. The dithiol moieties of Trx are reduced by receiving electrons from NADPH in the presence of TrxR. Reduced Trx in turn reduces proteins with disulfide bonds by transferring electrons from its reactive cysteines through thiol disulfide exchange reactions. Thus, NADPH, TrxR, Trx and thioredoxin interacting protein (TXNIP), the endogenous Trx inhibitor, are collectively termed the Trx system (Figure 6)57-60. There are three distinct forms of human Trx, encoded by separate genes; cytosolic Trx (Trx1), mitochondrial Trx (Trx2), and a Trx variant that is highly expressed in spermatozoa (SpTrx/Trx3).

The Thioredoxin-1 Gene

The gene encoding human Trx1 is located on chromosome 9 at bands 9q31. Its coding region spans over 13 kb and is organized in five exons separated by four introns. The Trx1 promoter contains two transcription start sites (Figure 7). Initial studies involving the Trx1 promoter region identified the first transcription start site (denoted as TSS1) at –110bp (with respect to the ATG start codon) by mapping the 5’ end of cDNA clone to a genomic sequence 83. Three Sp1 (specificity protein 1) binding sites were also located between –244bp and–183bp 83 and have been found to enhance transcription of the Trx1 gene 84. In contrast, a second transcription start site has been previously mapped (using primer extension analysis) to –74bp with a TATA box (TATAAA) located at –102bp 85. The TATA box is located downstream of TSS1 and is therefore only relevant for transcription initiation at TSS2. Luciferase assays, utilizing various wild-type and mutated Trx1 promoter fragments, revealed the roles for the ORE (oxidative stress responsive element), antioxidant response elements (ARE), three Sp1-binding sites and the TATA box in the activation of the Trx1 gene.

Truncated Thioredoxin (Trx80)

To date, only limited research has been performed on the biological roles of the truncated form of Trx (Trx80). Previously, it was termed eosinophil cytotoxicity-enhancing factor (ECEF) due to its eosinophil cytotoxicity and it has been first detected in the plasma of patients suffering from severe schistosomiasis 121-123. Trx80 (10 kDa) is a natural cleavage product of Trx1 sharing the 80 or 84 N-terminal amino acids with Trx1 124,125. It has been suggested that the enzyme responsible for its cleavage would be an inducible protease 126.Very recent findings, indicated that the disintegrins and metalloproteinases (ADAM10 and 17), two α-secretases processing the amyloid β precursor protein, are responsible for Trx80 generation in brain 127. Further work will have to establish other possible candidates in different tissues and under different pathophysiological conditions.
Macrophages are capable of cleaving full-length Trx1 to yield Trx80, which in contrast to the cytosolic localization of Trx1, is present mainly at the surface of monocytes 128. Trx80 is expressed also by U937 monocytes, cytotrophoblasts and CD4+ T cells 123,129. Recently, human brain samples and human primary cultures were shown to produce Trx80 and polymerize it into very stable aggregates migrating at approximately 30 kDa in SDS–PAGE 127.
Whereas the levels of Trx80 varied from 2 to 175 ng/ml, those of Trx1 varied from 16 to 55 ng/ml without any correlation to Trx80 concentrations, which increased significantly under inflammatory conditions 130,131. Trx80 activates monocytes and induces up-regulation of cell surface pathogen recognition receptors, molecules essential for T-cell activation and function 126 as well as release of proinflammatory cytokines evoking inflammation 90. Induction of human monocytes is associated with phenotypic cell changes 124, which differentiate into a novel line type named by Cortes-Bratti et al. Trx80-activated-monocytes (TAMs) with an intrinsic function contributing to the first line of defense against foreign intruder and triggering innate immunity 130. Thus, Trx80-activated monocytes could be more effective against bacteria and parasites via CD14 receptor up-regulation and other pathogen recognition receptors such as CD1 and the mannose receptor 128,130,131. Rheumatoid arthritis (RA) synoviocytes express the truncated form Trx80, and treatment with the pro-inflammatory cytokines IL-1β and/or TNF-α increases Trx80 cell expression playing an important role in the establishment and/or the development of RA autoimmunity. Lemarechal et al. reported that synoviocytes represent important target cells of Trx80, which respond in a concentration-dependent manner 126. Furthermore, in contrast to Trx1, Trx80 activates the classical and alternative pathways of complement activation through binding to complement initiators C1q and properdin, leading to production of the anaphylatoxin C5a 104.

The Thioredoxin Reductases

Thioredoxin reductase (TrxR), a homodimeric, relatively thermostable, selenium-containing flavoprotein oxidoreductase 106,132, catalyzes the NADPH-dependent reduction of Trxs disulfide and of numerous other oxidized cell constituents 133. TrxR is important for cell proliferation, antioxidant function, and redox signaling 134,135. These enzymes have three isoforms in mammals: TrxR1 in the cytosol, TrxR2 in mitochondria, and TrxR3 or TGR (thioredoxin glutathione reductase) present primarily in testes 136; in humans they are encoded by three different genes, TNXRD1, TNXRD2, and TNXRD3, respectively 133.
The C-terminal extension with the characteristic motif Gly-Cys-Se-Cys-Gly carrying the essential selenocysteine residue is unique for mammalian TrxRs and it forms a selenenylsulfide in the oxidized enzyme. Furthermore, this C-terminal extension enables TrxR to extend the electron transport chain from the catalytic disulfide to the enzyme surface and to prevent the enzyme from acting as a glutathione reductase by blocking the redox-active disulfide 137. Due to their flexible C-terminal tail, TrxRs have a broad range of substrates including glutaredoxin 2, protein disulfide isomerase, granulysin, and also some nonprotein substratessuch as selenite, dehydroascorbate, lipoic acid, ubiquinone, cytochrome C, or the cancer drugs motexafin gadolinium andalloxan 61,133.
Overexpression of TrxR1 has been observed in many tumors and cancer cells 2,136,138-141 rendering it an interesting target candidate for chemotherapy (discussed in chapter XIII). TrxR1 in cancer cells is essential for self-sufficiency of growth, progression into S phase 142, tumor progression, and metastasis 143. However, knockout of TrxR1 in mice exposed to a liver carcinogen showed a significantly increased incidence for chemically induced liver cancer 144. Thus, the dual role of TrxR in cancers, prevention/promotion, may depend on the stage of cancer development and on tissues as well.

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Thioredoxin Interacting Protein (TXNIP)

Human Trx-binding protein-2 (TBP-2)/vitamin D3 up-regulated protein 1(VDUP1)/Trx-interacting protein (TXNIP) is a negative regulator of Trx1 function because it interacts with the active center of Trx1, thereby inhibiting its reducing activity 145-148. It is one of six members of the mammalian α-arrestin family. The α-arrestins are structurally related to the β-arrestins, which are well-characterized mediators of G protein-coupled receptor signaling and endocytosis. TXNIP expression is ubiquitous and is induced by a variety of stresses, including UV light, γ-rays, heat shock, and H2O2, as well as glucose 149,150. TXNIP overexpression renders cells more susceptible to oxidative stress and promotes apoptosis 151-153. Furthermore, peroxisome proliferator-activated receptor gamma (PPARγ) activation stimulates apoptosis in human macrophages by altering the cellular redox balance via regulation of TXNIP 154. In addition to its inhibitory role, TXNIP plays important roles in lipid and glucose metabolism 70,155-158, inflammation 159-161, cardiac function 162, and carcinogenesis 163. Interestingly, the ability of TXNIP to inhibit glucose uptake was found to occur independent of Trx1 binding 164.

Regulation and Post-Translational Modifications of Thioredoxin-1

In spite of being cell cycle dependent, the expression of Trx1 can be upregulated by a variety of stress stimuli including hypoxia, Staph. aureus protein, lipopolysaccharide, O2, H2O2, phorbol ester, viral infection, photochemical oxidative stress, X-radiation, and UV irradiation 113,165. As mentioned earlier, Trx1 can undergo post-translational modifications. Glutathionylation of Trx1 leads to a reduction in the enzymatic activity of Trx1 under conditions of oxidative stress, which it seems to regain again, indicating the ability of Trx1 to degluthionylate itself by some means of autoactivation 112. S-Nitrosylation is the covalent addition of a NO moiety onto a cysteine thiol. It is a dynamic post-translational modification for the regulation of protein functions. S-nitrosylation of protein thiols may occur on exposure of specific redox-active motifs to NO, or as a result of transnitrosation/transfer reactions from low-mass carrier S-nitrosothiols or transfer from other protein S-nitrosothiols 166. Available evidences imply S-nitrosylation in cardiovascular, pulmonary, musculoskeletal and neurological (dys)functions, as well as in cancer (see 167 and 168 for more details). In contrast to other proteins such as caspases 169, peroxiredoxin 2 (PRX2) 170 or methionine adenosyl transferase 171, which are inhibited, the activity of Trx1is increased upon S-nitrosylation 114; consistently, purified Trx1 is sensitive to S-nitrosylation. Stimulation of HEK-293 cells with S-nitrosoglutathione resulted in Trx1 S-nitrosylation and consequently ASK-1 activation 172, whereas others found that S-nitrosylation occurring at Cys69 enables Trx1 to scavenge ROS, to preserve its redox regulatory activity, and to act as an antiapoptotic protein in endothelial cells 114. Interestingly, Trx system can act as a major S-nitrothiol–caspase-3 denitrosylating protein 173, because denitrosylation by Trx1 alone in the absence of TrxR1 was ineffective. Furthermore, Trx2 mediates Fas-induced denitrosylation of mitochondria-associated S-nitrothiol–caspase-3 and promotes apoptotic signaling 168,173. Cys73 of Trx1was shown to be responsible for the transnitrosylation of caspase 3 at Cys163 because mutation of Cys73 to Ser, yielded a mutant exhibiting normal disulfide reduction activity, which was resistant to nitrosylation, and which lacked transnitrosylation activity toward caspase 3 117. On the other hand, the redox states of the Cys32 and Cys35 seem to be crucial regulators determining the nitrosylation of Trx1 at Cys73 and its ability to transnitrosylate target proteins 119,138. Therefore, under certain conditions, Trx1 plays a major role in protein S-denitrosylation and it can also trans-S-nitrosylate other proteins 138,168,174.

General Functions of Trx

Since 1964, the number of publications on thioredoxin is exponentially growing (Fugyre 9) representing a promising molecule. The Trx system is a crucial and essential system for the protection against oxidative stress, for the maintenance of the cellular redox balance, and for the regulation of differentiation and cell fate (recently reviewed 175). This wide range of cellular functions of Trx system leads into its involvement in a variety of diseases (Figure 9) 176.

Table of contents :

Introduction
1. Cardiovascular diseases (CVDs)- the main cause of death in europe
1.1. Atherosclerosis – a chronic inflammatory disease
1.2. Lipoproteins- role of oxidized LDL in atherosclerosis
2. Macrophage polarization- a response to the microenvironment
3. Oxidative Stress – the underlying cause of many disease
4. The thioredoxin system
4.1. The thioredoxin-1 gene
4.2. Structure of thioredoxin-1
4.3. Truncated thioredoxin (Trx80)
4.4. The thioredoxin reductases
4.5. Thioredoxin interacting protein (TXNIP)
4.6. Regulation and post-translational modifications of thioredoxin-1
4.7. General functions of Trx1
4.8. Role of Trx1 in apoptosis
4.9. Trx regulates glucocorticoid and estrogen receptors
4.10. The Trx system and Aging
4.11. The Trx system in cardiovascular diseases
5.Context of the project
Results and discussion
6. Thioredoxin-1 induces M2 polarization
6.1. Study presentation
6.2. Article I
7. Truncated thioredoxin induces M1 polarization
7.1. Study presentation
7.2. Article II
8. Possible signaling pathway involved in macrophage polarization
8.1. Study presentation
8.2. Regulation of cell signaling by Trx1
8.2.1. Extracellular Trx1
8.2.2. Cytoplasmic Trx1
8.2.2.1. Mitogen-activated protein kinases
8.2.2.2. Calcium signaling
8.2.3. Nuclear Trx1
8.3. The mTOR signaling pathway
8.4. Materials and methods
8.4.1. Extracellular Trx1 Isolation and treatment of mouse peritoneal macrophages
8.4.2. Western Blot Analysis
8.5. Results
8.5.1. Both Trx1 and Trx80 activate Akt
8.5.2. Trx80, but not Trx1, activates mTOR
8.5.3. Inhibition of mTOR downregulated the expression of inflammatory cytokines
8.5.4. Trx80 activates mTOR in dose-dependent manner
8.5.5. mTOR inhibition orientes resting macrophages toward M2 phenotypes
8.6. Discussion
General Discussion
Conclusions
References

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