Extracellular Trx1 Isolation and treatment of mouse peritoneal macrophages

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Trx Regulates Glucocorticoid and Estrogen Receptors

Glucocorticoids, as major peripheral effector of the hypothalamic-pituitary-adrenal axis, play an essential role in reestablishing the homeostatic status in every peripheral tissue in human., Upon binding of glucocorticoids to their receptor, they promote the dissociation of heat shock proteins, and the ligand–receptor complex translocates to the nucleus, where it binds to palindromic DNA sequences, The redox status of cysteine residues of glucocrticoids maintain their structure and binding abilities 199,200. Previously it has been revealed that Trx1 is required for generating the ligand-binding conformation of the glucocorticoid receptor. Antibody-mediated sequestration of either Trx or TrxR inhibited the ligand binding activity of the glucocorticoid receptor 201. The same group conducted another study to show the direct effect of Trx1 on the DNA binding of the gluccocorticoid receptor. They have inhibited the binding ability of receptors by methyl methanethiosulfonate (MMTS), which can be reversed by adding dithiothreitol (DTT). Surprisingly, the promoting effect of partially purified Trx1 on DNA binding of MMTS-treated receptors in the presence of DTT was not affected by anti-Trx1 serum 202.
Additional studies on the role of Trx1 on the responsiveness to glucocorticoids revealed that the downregulation of Trx1 by antisense ODN or treatment with H2O2 negatively modulated the glucocorticoid receptor function and decreased the glucocorticoid-inducible gene expression. However, the cellular responsiveness to glucocorticoids was rescued by overexpression of Trx1 103,203.

The Trx System in Cardiovascular Diseases

Growing evidence indicates that overproduction of ROS under pathophysiological conditions is an integral component in the development of CVD such as atherosclerosis, ischemic heart disease, hypertension, cardiomyopathies, cardiac hypertrophy and congestive heart failure 54,55 because they activate various signaling pathways that underlie the vascular inflammation in atherogenesis (Figure 10). Therefore, Trx1 may play a beneficial role antagonizing CVD. In isolated perfused hearts adapted to ischemia and reperfusion by short cycles of ischemia and reperfusion (I/R), the protection is apparently afforded by the enhanced induction of Trx1, because ROS increases, when Trx1 is inhibited by a suitable inhibitor 216. Further studies conducted in animal models in vivo suggest that Trx1 has many beneficial functions in the heart 59,193. Only recently, it has been demonstrated that impaired angiogenesis and myocardial dysfunction can be counteracted by adenoviral vector-based gene therapy encoding Trx1 155. Several studies indicate that Trx1 plays a role in the pathogenesis of atherosclerosis. NO and peroxynitrite contribute to the damage to smooth muscle and endothelial cells seen in atherosclerotic plaques 217 with increased Trx and TrxR mRNAs 218. Trx1 prevents the NO-dependent inhibition of purified NO-synthase. Furthermore, endogenous levels of Trx1 are critical for restoring the NO-induced loss of eNOS activity because overexpression of Trx1 prevents the NO-induced loss of eNOS catalytic activity (Figure 10) 96,219. It has been well documented that the mechanism(s) responsible for the improved angiogenesis as well as cardioprotection mediated by resveratrol, operates both in vitro and in vivo through the induction of vascular endothelial growth factor expression triggered by Trx1 and HO-1. Significant increase in the Trx1 expression was observed at baseline level in the myocardium of streptozotocin-induced diabetic rats after I/R. A protective role for Trx1 has also been implicated in cardiac hypertrophy. Several studies support the concept that Trx1, as antioxidant, inhibits and protects from cardiac hypertrophy in animal models. Transgenic mice with a cardiac-specific overexpression of a dominant negative Trx1 mutant showed a loss of the endogenous oxidoreductase activity of Trx1 219-222. In patients with coronary artery disease (CAD), high homocysteine levels may cause low Trx1 activity, which is closely correlated to the extent and severity of the CAD 119. During I/R, Trx1 also inhibits expression of proinflammatory cytokines, chemotaxis, complement activation, and neutrophil adhesion. This is an important issue, because I/R stimulate cell adhesion molecules and the migration of neutrophils into myocardial tissue. Subsequent production of inflammatory cytokines and ROS enhances myocardial injury. Another potentially important mechanism is the effect of Trx1 upon ion channel remodeling. Downregulation of the K+ channel by oxidative stress could cause lethal arrhythmia and cardiac contractile dysfunction. Upregulation of Trx1 may prevent downregulation of the Kv4 channel and the subsequent electrical instability during I/R 59. Additionally, exogenous Trx1 exerts distinct cytoprotective effects on cerebral I/R injury in mice by means of its redox-regulating activity 223. Furthermore, it has been demonstrated that S-nitrosation of Trx1 potentiates its protective activity against myocardial I/R 115. It has been suggested that there is a possible association between Trx1 secretion and the severity of heart failure. Trx1 is increased in both inflammatory cells and myocytes during myocarditis 111. Furthermore, increased Trx1 levels have been observed in several oxidative stress-associated CVDs, for instance, serum Trx1 levels were significantly increased in patients with abdominal aortic aneurysm (AAA) relative to healthy subjects. Indeed, levels correlate well with the size and expansion of AAA, suggesting its potential role as a biomarker of AAA evolution 224. Furthermore, recent analysis of TXNIP-/- mice revealed suppression of genes, which participate in mitochondrial metabolism 225. Consequently, TXNIP-/- mitochondria were functionally and structurally altered, showing reduced oxygen consumption and ultrastructural derangements 225. TXNIP deletion would therefore enhance I/R damage. Surprisingly, TXNIP-/- hearts had greater recovery of cardiac function after an I/R insult 225. Similarly, cardiomyocyte-specific TXNIP deletion reduced infarct size after reversible coronary ligation. Thus, in addition to reduced mitochondrial function, deletion of TXNIP enhanced anaerobic glycolysis 225. Whereas mitochondrial ATP synthesis was minimally decreased by TXNIP ablation, cellular ATP content and lactate formation were higher in TXNIP-/- hearts after I/R injury 225. Inhibition of glycolytic metabolism abolished the protection of the heart by TXNIP deficiency under hypoxic conditions. Thus, although TXNIP deletion suppresses mitochondrial function, protection from myocardial ischemia is increased as a result from a coordinated shift to enhanced anaerobic metabolism, which supplies an energy source outside mitochondria 225.

Truncated Thioredoxin induces M1 polarization

Truncated thioredoxin, Trx80, is a natural cleavage product of Trx1with 10 kD 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. Up to date, there are very limited works on Trx80. Previously, it has been shown that macrophages can cleave Trx1 producing Trx80 128 which can 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. Since Trx80 has very different properties from the full-length protein, lacking the disulfide reductase activity of the full-length species, and instead having pro-inflammatory cytokine-like effects on immune cells, 124,131 we further investigated the vascular proinflammatory mechanism to clarify its atherogenic implication 48. In his study, we report that Trx80 on one hand potentiates the expression of M1 macrophages markers, through the AP-1 and Ref-1 transcription factors-independent pathway and on other and it blunts the expression of anti-inflammatory cytokines. Taken together, these studies indicate that Trx80 functions as regulator of macrophage phenotype tipping the balance toward the pro-inflammatory M1 state. As a consequence, atherosclerotic plaques become larger and probably more unstable.

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Possible signaling pathway involved in macrophage polarization

The importance of redox signaling is increasingly recognized, despite the highly transient and volatile nature of the redox modifications that makes them very difficult to access for broad in depth investigations. The various redox modifications are highly target- and site specific, and the Trx, Grx, and Prx systems are pivotal players in redox signal transduction both as transducers and as regulators of second messenger levels. As in previous two studies, we have shown that Trx1 and Trx80 play different role in macrophage polarization and the development of atherosclerosis. Therefore, it is thought that each may use different signaling pathway or regulate the same signaling pathway but in different way. Among the candidate signaling pathways, Akt pathway is most likely to be used by Trx1 and/or Trx80.
Akt (also known as PKB) is a family of three serine/threonine protein kinases (Akt1, Akt2, and Akt3) that regulate a host of cellular functions, including cell survival, proliferation, differentiation, and intermediary metabolism 240. In the vascular wall, Akt has been shown to play an important role in the proliferation and migration of endothelial cells, regulation of vascular permeability, and angiogenesis 241-243. Recent studies of Akt knockout mice have shown that despite significant sequence homology, the three Akt isoforms have some nonredundant functions. Although Akt1-deficient mice exhibit overall growth impairment 244, Akt2 knockout mice have impaired glucose tolerance and insulin resistance 245, and Akt3 nulls display a selective reduction in brain size 246. Additionally, it has been reported that a global absence of Akt1 in vivo enhances atherosclerotic lesion burden and promotes coronary atherosclerosis in a mouse model of atherosclerosis suggesting that Akt1 exerts vascular protection against atherogenesis 240 interestingly it has been demonstrated that Akt2 ablation results in the M2 polarization of macrophages whereas Akt1 ablation promotes their M1 polarization 247.Therefore, the implication of Akt pathway has been studied to explore the signaling pathway used by Trx1 and/or Trx80 to exert their effects.

Regulation of Cell Signaling by Trx1

Depending on its subcellular localization, Trx1 exerts different roles. It can be found in the extracellular environment, on the cell-surface and intracellularly.Trx1shows redox regulatory functions in signal transduction and transcriptional mechanisms.

Extracellular Trx1

In spite of lacking a signal peptide, Trx1 can be secreted by cells through an unknown mechanism 248,249. Extracellular Trx1, either secreted or exogenously added, triggers a variety of physiologic or pathologic functions. Until now, no binding of Trx1 to a specific cell-surface receptor has been characterized. It seems that Trx1 acts as an autocrine growth factor and in synergy with other cytokines as a potent costimulatory molecule from the outside of cells 250. Although it has not yet been extensively studied, Trx1 has been reported as a lipid raft (LR)-associated protein. LR are specialized domains of the plasma membrane that cluster specific proteins and provide a dynamic scaffold for organizing cellular processes such as signal transduction (Figure 11) 251. There is convincing evidence that LRs redox signaling platforms may mediate the actions of Trx1 on leukocyte–endothelial cell interaction related to redox regulation during inflammation 252. On the other hand, LR clusters get endocytosed and form redoxosomes that mainly produce ROS as signaling molecules to evoke intracellular downstream responses. Trx1 can be internalized into the cells through LR-mediated endocytosis 253. Increasing evidence suggests that LR redox signaling may contribute to infection, host defense, and to the development of different diseases such as atherosclerosis, obesity or metabolic syndrome and tumor progression 254. Further studies are needed to investigate the molecular mechanisms involved in the formation of this membrane signaling complex highlighting the possible role of Trx1 through these platforms.

Table of contents :

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 diseases
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 Mitogen-activated protein kinases 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
List of Contents
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


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