The A to Z of Modulated Cell Patterning by Mammalian Thioredoxin Reductases

Abstract

Mammalian thioredoxin reductases (TrxRs) are selenocysteine-containing proteins (selenoproteins) that propel a large number of functions through reduction of several substrates including the active site disulfide of thioredoxins (Trxs). Well-known enzymatic systems that in turn are supported by Trxs and TrxRs include deoxyribonucleotide synthesis through ribonucleotide reductase, antioxidant defense through peroxiredoxins and methionine sulfoxide reductases, and redox modulation of a number of transcription factors. Although these functions may be essential for cells due to crucial roles in maintenance of cell viability and proliferation, findings during the last decade reveal that mammals have major redundancy in their cellular reductive systems. The synthesis of glutathione (GSH) and reductive functions of GSH-dependent pathways typically act in parallel with Trx-dependent pathways, with only one of these systems often being sufficient to support viability. Importantly, this does not imply that a modulation of the Trx system will remain without consequences, even when GSH-dependent pathways remain functional. As suggested by several recent findings, the Trx system in general and the TrxRs in particular, function as key regulators of signaling pathways. In this review article we will discuss findings that collectively suggest that modulation in mammalian systems of cytosolic TrxR1 (TXNRD1) or mitochondrial TrxR2 (TXNRD2) influence cell patterning and cellular stress responses. Effects of lower activities include increased adipogenesis, insulin responsiveness, glycogen accumulation, hyperproliferation, and distorted embryonic development, while increased activities correlate with decreased proliferation and extended lifespan, as well as worse cancer prognosis. The molecular mechanisms that underlie these diverse effects, involving regulation of protein phosphorylation cascades and of key transcription factors that guide cellular differentiation pathways, will be discussed. We conclude that the selenium-dependent oxidoreductases TrxR1 and TrxR2 should be considered as key components of signaling pathways that control cell differentiation and cellular stress responses.

Graphical abstract

Cellular patterning programs and redox control

Redox regulation, i.e. regulatory oxidation-reduction modification of key proteins in cellular signaling pathways, is fundamental to life. Many diseases arise from imbalances in redox processes, including events of oxidative stress, hypoxia and ischemia-reperfusion injury. Importantly, redox processes are also essential parts of normal physiological events [1]. The two major reductive systems in mammalian cells are the thioredoxin (Trx) and glutathione (GSH) systems, which are generally complementary to each other in life supporting functions, but may differ in the context of modulated redox signaling pathways [2–7]. Indeed, these systems are well known to be able to modulate a large number of signaling pathways in the cytosol, nuclei, and mitochondria [4, 8–19], with major effects in cellular pathways of reduction and redox control [14, 19, 20].

Early insights suggesting both a robust redundancy between cytosolic disulfide reductase systems and their possible additional signaling roles in higher eukaryotes came with the onset of mouse genetic models having disruptions in these systems. First it was noted that germline genetic ablation of glutathione reductase (Gsr) had almost no measurable phenotypic impact on mice, and very curiously also did not substantially affect either intracellular levels or redox state of hepatic glutathione in the homozygous mutant animals, i.e. only a minor increase in the already very low intracellular glutathione disulfide levels were detected [21, 22]. This later observation suggested that mammalian cells had alternative means of sustaining pools of reduced GSH. These inferred systems were not resolved for more than another decade, after which it was revealed that both methionine-dependent de novo synthesis of GSH and cross-trafficking of reducing power from the cytosolic TrxR1/Trx1 system can participate in sustaining reduced GSH pools in the absence of Gsr [2, 23]. Further suggesting the GSH system might be of only minor importance in cell and organismal homeostasis, it had already been well established that mammalian cells as well as adult animals and humans were highly tolerant of chronic severe systemic GSH depletion by the drug buthionine sulfoximine (BSO) [24–28].

In contrast to these observations on the GSH system, homozygous germline knockouts of the genes encoding Trx1, Trx2, TrxR1, or TrxR2 were all found to result in embryonic lethality, suggesting that the cytosolic and mitochondrial Trx systems, unlike the GSH system, are critical for basal homeostasis in mammalian cells, at least during embryogenesis [29–32]. Interestingly however, none of the mouse thioredoxin system-knockout models showed simple zygotic arrest, as one might have expected for activities critical to basal cell survival or DNA replication. Rather, each homozygous mutation sustained early development and extensive cell proliferation, followed by a later lethal crisis. More detailed analyses of mouse embryos lacking TrxR1 verified that early TrxR1-null embryos underwent robust proliferative expansion and differentiation of early tissues, including trophectoderm and primitive endoderm, but arrested prior to gastrulation [33]. Single embryo transcriptome profiling and marker analyses revealed that mesodermal genes were not expressed and, indeed, there was no evidence of formation of either node or primitive streak in the mutant embryos [33]. These observations suggested that TrxR1-null embryos had no major basal cellular deficiencies, but rather had a patterning defect in which the intercellular signaling that establishes early embryonic patterning was affected [33].

The apparent normal proliferation and development of TrxR1-null embryonic trophectoderm and primitive endoderm was intriguing. One of our laboratories initiated a study in which a Cre-responsive conditional-null allele of the Txnrd1 gene (encoding TrxR1) was combined with a Cre-responsive dual-fluorescent reporter gene and inducible Cre expression systems in an attempt to use marked-mosaic analyses to identify cell types in vivo that absolutely required TrxR1 expression. We generated mosaic animals with marked TrxR1-null cells distributed throughout all organs and tissues of the fetal, juvenile, or adult body, and yet we were unable to identify any cell type that did not tolerate long-term disruption of TrxR1 (EES, unpublished data). To provide a convenient model system for studying the in vivo functions of TrxR1 in animals, we adopted a liver-specific model based on disruption of the conditional-null Txnrd1 allele coincident with hepatocyte differentiation using the classical AlbCre transgene [34, 35]. Ours and other groups also developed models lacking TrxR1 in different cell types including fibroblasts, neurons, heart, adipocytes, various cancer cells, and others [29, 32, 36–38]. Detailed studies on the TrxR1-null livers revealed that basal cell functions, including robust developmental and regenerative proliferation, were indeed sustained in the absence of TrxR1 [39, 40]. Intriguingly, the TrxR1-null livers also showed no measurable markers of oxidative stress, yet transcriptome analyses on these livers indicated that they had strong chronic induction of the Nrf2 oxidative stress response pathway [36, 41]. Similar findings were subsequently found using pharmacologic inhibition of TrxR1 [42]. These results suggested that TrxR1 might be a regulatory component on a signaling pathway that controls Nrf2 [10, 43]. In combination with the patterning defects in TrxR1-null embryos, these findings hinted that TrxR1 might play roles in modulating intracellular aspects of various signaling pathways.

In this article we shall specifically survey and discuss how the mammalian NADPH-dependent selenoprotein thioredoxin reductases TrxR1 and TrxR2 seem to modulate a large number of cellular processes that are important in regulation of cell patterning.

Mammalian thioredoxin reductases

Background and overall functions

The whole Trx system is NADPH dependent since it is propelled by NADPH-dependent oxidoreductases, mainly cytosolic TrxR1 (encoded by TXNRD1) and mitochondrial TrxR2 (encoded by TXNRD2). The system is also selenium-dependent since these enzymes are selenoproteins, i.e. they have a catalytic selenocysteine residue, and they are furthermore subject to splicing that results in variants of the enzymes differing at their N-terminal domains [14, 44–47]. It should be noted, considering cellular signaling pathways, that the normally cytosolic TrxR1 enzyme can also be found in the nucleus in the alternative splice-form of TXNRD1_v2, where it can modulate transcription factor activities, such as the estrogen receptors [14, 46]. The alternative splice-form of TXNRD1_v3 is targeted to cell membranes, where it co-localizes with membrane rafts and has the capacity to promote formation of filopodia [14, 48–50]. These splice variants of TrxR1 have not yet been examined at depth in relation to cellular signaling pathways, but should clearly also be specifically considered in this context. It should also be noted that mammalian TrxRs display very broad substrate specificities and reduce many low molecular weight compounds in addition to their primary Trx substrates [14]. Enzymatic activities with certain regulatory low molecular weight metabolites may hence, in theory, also be related to the impact of TrxR status on cellular patterning programs, but this possibility has yet been insufficiently studied.

Thioredoxins as primary TrxR substrates

The primary substrates of TrxR1 and TrxR2 are Trx1 and Trx2, respectively, and most actions of the mammalian thioredoxin system are likely to be carried out by the activities of Trx1 and Trx2 [12, 14, 51–55]. However, additional substrate proteins for TrxR1 such as the Trx-related protein of 14 kDa (TRP14, TXNDC17) are also likely to be important in relation to signaling events [56–61]. It should furthermore be noted that although major signaling aberrations can be seen upon genetic deletion of TrxR1 (the Txnrd1 gene in mice) or TrxR2 (the Txnrd2 gene in mice) these are not necessarily related to effects on Trx1 or Trx2 activities, respectively, because of significant crosstalk in redox pathways and specific effects on signaling depending upon more than solely general impairments of the cellular reduction capacity [8, 37, 40, 41, 62].

Ribonucleotide reductase

The antioxidant and redox-regulatory functions of the Trx system support cell growth and viability through a diverse set of mechanisms. Ribonucletide reductase, that catalyzes and controls the synthesis of DNA precursors, is an example of an important enzyme that receives reducing equivalents from the Trx system to maintain its function, although it can also be reduced by glutaredoxins [63–65], as in bacteria [66, 67]. Several redox active proteins can thus support ribonucleotide reductase, including Grx2 and its different splice variants [68]. Either TrxR or GSH can in turn reactivate Grx2 through reduction [64, 69, 70]. This redundancy in support of ribonucleotide reductase activity likely helps to explain the non-coherent expression and localization patterns of ribonucleotide reductase, Trx and TrxR in mammalian cells and tissues [71–73].

Peroxiredoxins

The peroxiredoxins (Prxs) are important antioxidant proteins that exist in different cellular compartments in high abundance. Their antioxidant functions and catalytic activities are in turn dependent upon direct reduction by Trxs. Prxs efficiently reduce H₂O₂ and are considered to be important antioxidant enzymes and also modulators of intracellular signaling through regulation of oxidative bursts [15, 18]. Inhibition of TrxR through compounds such as auranofin or isothiocyanates, has identified the mitochondrial Prx3 to be most susceptible peroxiredoxin to oxidation; time dependent analysis of the rate of oxidation during low dose TrxR1 inhibition showed that Prx3 was affected to a greater extent than cytosolic peroxiredoxins [74, 75]. The antioxidant function of Prx3, in the mitochondria, is supported by Trx2 and TrxR2 as well as a GSH-dependent peroxidase activity of Grx2 [76]. If Prx3 becomes overoxidized to its sulfinic acid form, the Trx system can no longer reactivate Prx3 but instead sulfiredoxin is required, which under such conditions becomes translocated from its natural location in the cytosol into the mitochondria [77]. The events of Prx3 overoxidation and sulfiredoxin being targeted to mitochondria are, interestingly, directly linked to the cellular mechanisms of circadian rhythm [78–80]. This poses the question whether activities or functions of the core players of the Trx system can also be directly linked with circadian rhythm. Some observations in Drosophila melanogaster [81] and plants [82] suggest this may be the case, but further studies of this topic are clearly needed.

Methionine sulfoxide reductases

The important Trx-dependent methionine sulfoxide reductase (Msr) enzymes repair methionine sulfoxide residues in proteins by reduction. Two MsrA and MsrB isoenzymes are specific for reduction of either methionine-S-sulfoxide or methionine-R-sulfoxide. Msrs are found in many organisms and, like TrxRs, some but not all Msrs are selenoproteins [83, 84]. In mammals, MsRB1 together with Mical enzymes controls actin polymerization through regulated methionine oxidation status [85]. Impaired Msr functions are related to degenerative disease processes and aging [83, 84]. For example, expression levels of both variants of mammalian methionine sulfoxide reductases decreased with increased age in rats, and also cytosolic Trx1 and TrxR1, which reduce Msrs, were found to be decreased [86]. Genetic deletion of MsrA increases oxidative stress, resulting in neurological defects and a reduced lifespan [87]. Selenium deficiency is known to decrease TrxR activity and combined with MsrA deficiency an increased oxidative damage is detected [88].

Effects of modulated activities of thioredoxin reductases on cellular patterning in mammalian cells and tissues

A large number of observations in recent years indicate that modulation of TrxR isoenzymes is typically linked with altered cellular phenotypes, with in many instances strong indications of casual relationships. In Table 1 and Figure 1 we have summarized an “A to Z” of a number of key observations in line with this notion, which shall here be discussed in further detail.

Table 1. The A to Z of Modulated Cell Patterning by Mammalian Thioredoxin Reductases.

This table lists selected key references from the literature that illustrate the effects of modulated expression of TrxR1 or TrxR2 on cell patterning. For a schematic illustration of these effects, see Figure 1 with the index letters as references.

Index	Aberration	Cellular system	Effects on cell patterning	Comment	Ref.
	Forced Overexpression of TrxR1
A		TrxR1 overexpression in Bovine arterial endothelial cells	TrxR1 overexpression is induced by Nrf2 activation and can enhancee TNFα-induced DNA binding activity of NF-κB and dependent gene expression		[206, 218]
B		TrxR1 and TrxR2 overexpression in HEK293 cells	Decreased cell proliferation and stimulation of differentiation	Expression of cell differentiation markers CK18, CK-Cam5.21 and BerEP4, decrease in GR activity and expression	[219]
	Forced Overexpression of TrxR2
B		TrxR1 and TrxR2 overexpression in HEK293 cells	Decreased cell proliferation and stimulation of differentiation	Expression of cell differentiation markers CK18, CK-Cam5.21 and BerEP4, decrease in GR activity and expression	[219]
C		TrxR2 overexpression in Neuro2A, COS-7 and HeLa cells	Decreased growth rate		[220]
D		TrxR2A (alternative splicing variant) overexpression in HeLa and HEK293 cells	Expression induced apoptosis, decrease in cell proliferation of stable HEK293 cell lines.	Cleavage of procaspase-3 and PARP	[221]
	Endogenous Upregulation of TrxR1
E		3T3-L1	Differention of 3T3-L1 cells into adipocytes	Upregulation of TrxR1, Trx1, TrxR2 and Trx2 during differentiation	[105]
F		Alternative splicing of TrxR1 in HEK293 cells	Regulation of ER signaling	Modulation of estrogen signaling	[46]
G		Upregulation of TrxR1 in cancer	Upregulation of TrxR1 transcripts as well as activity correlates with worse cancer prognosis	Analyses of human data	[98–100]
	Endogenous Upregulation of TrxR2
E		3T3-L1	Differention of 3T3-L1 cells into adipocytes	Upregulation of TrxR1, Trx1, TrxR2 and Trx2 during differentiation	[105]
H		Several model organisms	Overexpression of TrxR2 correlates with longer lifespan	Upregulation of TXNRD2 transcript and activity seen in long- lived long-lived species of rodents, primates, and	[106]
	Endogenous Downregulation of TrxR1
I		miR-23a and miR-23b dependent regulation of TrxR1 expression in C2C12 mouse myoblasts	miR-23a and miR-23b control skeletal myogenic differentiation through regulation of TrxR1 expression	Decreased expression of TrxR1 induced differentiation of C2C12 myoblasts and muscle satellite cells. Overexpression of TrxR1 delayed C2C12 myoblasts differentiation	[107]
J		TrxR1 expression in a human patient study	TrxR1 and TrxR1- v.2,3,5 correlation with differentiation	TrxR1 and TrxR1-v.2,3,5 expression was correlated with differentiation in squamous carcinoma.	[108]
K		Naturally occurring mutation in human patients	A homozygous Pro190Leu mutation correlates with generalized epilepsy	Found by whole-exome sequencing in a family with genetic generalized epilepsy	[109]
	Endogenous Downregulation of TrxR2
L		Rare mutations in the FAD-binding domain of the TNXRD2 gene (A59T and G375R) of human patients with dilated cardiomyopathy (DCM)	Heterozygous carriers of the two mutations mutations displayed impaired left ventricle function	However these mutations only contribute to disease burden since other DCM causing mutations have been described	[110]
	Knockout of TrxR1
M		Whole animal mouse model	No mesoderm formed. Disruption of gastrulation	No evidence of increased oxidative stress	[33]
N		MEF −/−	Increased differentiation, lipogenesis	Strong effects on Akt signaling; possible to block with NAC	[37]
O		MEF −/−	Increased apoptosis	TrxR1 is required for clearance of glucose derived H2O2	[38]
P		Hepatocyte specific disruption of Txnrd1	Metabolic switch from lipogenic genes towards glycolysis, increased proliferation and hepatomegaly	Txnrd1-null livers were resistant to APAP- induced hepatotoxicity, replication is dependent on either at least one allele of Txnrd1 or sufficient GSH levels, and triple knockout of Txnrd1, Trx1 and GR yields pronounced hepatomegaly	[62, 111–113]
Q		Neuronal specific Txnrd1 conditional knockout	Morphological alterations	Reduced proliferation of granule neurons resulting in thinner EGL in mutants (Postnatal day 7). Reduced weight, severe ataxia. Strongly disorganized development in anterior lobules (I–VI). Cereberal hypoplasia. Radial glial cells or granule cell precursors likely to be affected	[114]
R		MEF TrxR1 −/−	Increase in PDGF induced proliferation	TrxR1 deficient cells display an increase in PTP1B oxidation corresponding to a decrease in activity and increase in PDGF induced proliferation	[58]
	Knockout of TrxR2
S		Whole mouse Txnrd2 knockout	Embryonic lethal at day 13	Embryos were smaller and showed severe anemia. Apoptosis was increased in liver. Decreased proliferation of cardiomyocytes. Cardiac specific ablation of TrxR2 resulted in cardiomyopathy	[115]
T		Heart specific Txnrd2 knockout	Increased heart to body weight, mitochondrial degeneration	Increased autophagy, reduced blood pressure, stabilization of HIF, decreased oxygen consumption and increased ROS	[116]
U		MEF −/− subcutaneous tumor model	Decreased vascularization	Delay of angiogenic switch, impaired tumor angiogenesis. Increased prolyl hydroxylase (PHD2) levels with corresponding decrease in Hif1α. Decrease in VEGF-A levels. Increase in JNK activation	[121]
V		Cardiomyocyte specific Txnrd1- or Txnrd2 deficient mice		Increased vulnerability to I/R induced injury. Increased mitochondrial ROS	[117]
X		Conditional B- and T- cell- specific inactivation of Txnrd2	No effect on proliferation		[118]
Y		TrxR2 shRNA knockdown in Chondrogenic ATDC5 cells	TrxR2 deficiency enhance differentiation and apoptosis	Knockdown stimulates expression of Cartilage glycosaminoglycans (GAGs) and expression of (ECM) genes Collagen 2 and Aggrecan. Activation of Akt signaling and chondrocyte proliferation	[119]
Z		Endothelium specific Txnrd2 knockout mice subjected to artery ligation	Impaired vascular remodeling processes in the model of femoral artery ligation	Endothelial deletion had no change in phenotype under normal conditions. However, artery ligation lead to increased vascular stiffness. Impaired EC vasodilation. The presence of global thrombi was seen in Txndr2 knockout animals suggesting a proaterogenic phenotype. Proinflammatory phenotype. Increase in leukocyte transmigration into tissues. Impaired sprouting of Txnrd2 KO cells in vitro.	[120]

Figure 1. Summary of Modulated Cell Patterning Effects Linked to Mammalian Thioredoxin Reductases.

This figure schematically summarizes key effects of forced overexpression, endogenous upregulation, endogenous downregulation or knockout of either TrxR1 or TrxR2 in cells, mice or human, as indicated. For further details and references, see the text and Table 1. The one-letter indices refer to the corresponding indices in Table 1.

Forced overexpression

With regards to cellular phenotypes triggered by forced overexpression of TrxR1 or TrxR2 it should be noted that because of highly inefficient and demanding synthesis machineries required for expression of selenoproteins in mammalian cells [89, 90], it is a difficult task to overexpress these proteins. Moreover, if the selenocysteine (Sec)-encoding UGA codon in the TXNRD1 or TXNRD2 transcripts leads to translational termination, instead of Sec insertion, the enzymes will be expressed as two-amino acid truncated forms ending with –Gly-Cys instead of their natural C-terminal end of –Gly-Cys-Sec-Gly. Such truncated forms of the enzymes are not inertly inactive, but interestingly retain redox activities as NADPH oxidases with pro-oxidant redox cycling capacity coupled to low molecular weight substrates, such as several quinone compounds including juglone. This pro-oxidant activity can become toxic to cells and these shortened forms of the enzyme were named SecTRAPs, for Selenium compromised thioredoxin reductase-derived apoptototic proteins [10, 91–95]. Formation of SecTRAPs in cancer cells during chemotherapy with alkylating agents can be beneficial in terms of therapeutic effects, while at least lower levels of SecTRAPs formed in normal cells may possibly trigger oxidative signaling pathways. Thus, the phenotypes observed upon forced expression of TrxR1 or TrxR2 may in fact be due to increased oxidative stress rather than increased reductive power, which may possibly explain some of the apparently paradoxical effects seen in such model systems [8]. In Table 1 and Figure 1 a number of observations are listed with effects upon cell proliferation after forced overexpression of either TrxR1 and TrxR2, which may hence possibly at least in part be due to increases in oxidative stress due to SecTRAP formation.

Endogenous overexpression

Several growth conditions and cell patterning events are related with endogenously increased expression levels of TrxR1 or TrxR2. The expression of TrxR1 is, interestingly, guided by a typical housekeeping-type promoter in combination with AU-rich elements in the mRNA that guide regulated mRNA stability, otherwise typically found in signaling proteins [19, 96]. Responding to oxidative stress, the TrxR1 transcript is also upregulated through Nrf2 [97] and the enzyme is highly overexpressed in several forms of cancer, where its high overexpression moreover correlates with poor prognosis [98–100]. Both the TXNRD1 and TXNRD2 genes furthermore give rise to several different splice variants, encoding TrxR1 or TrxR2 variants differing at their N-terminal ends, some of which may be dedicated players in cellular signaling pathways [44–49, 101, 102]. One of the variants of TrxR1, TXNRD1_v2, is targeted to the nucleus where it can modulate estrogen signaling by interactions with the estrogen receptor [46]. Another variant, TXNRD1_v3, is myristoylated and targeted to the cell membrane where it can trigger formation of filopodia and is possibly involved in receptor-linked or cell-to-cell mediated signaling events [103, 104]. The existence of these splice variants illustrates well that sole expression levels of TrxR1 (and its variants) can not be the only determinants of the effects of TrxR1 on cell patterning, but also compartmentalization effects and dedicated functions of specific splice variants. Such details in the mechanisms of TXNRD1- or TXNRD2-linked expression levels in relation to specific phenotypes are often overlooked, but should be considered for a full understanding of molecular mechanisms leading to any observed effects. Notwithstanding these aspects, it was found that both TrxR1 and TrxR2, as well as Trx1 and Trx2, are endogenously upregulated when 3T3-L1 cells differentiate into adipocytes [105]. This was an interesting finding since it was also shown that upon knockout of Txnrd1 in mouse embryonic fibroblasts this strongly promoted adipogenesis, which occurred in conjunction with potently augmented Akt and PPARγ activities [37].

With regards to endogenous upregulation of TrxR2 this seems to correlate with longer lifespan in several different model organisms [106]. The molecular mechanisms that may explain this interesting correlation are yet unknown.

Endogenous downregulation

Examples of studies correlating endogenous downregulation of TrxR1 or TrxR2 with cell patterning phenotypes are scarce. Recently one interesting study however discovered a control of TrxR1 expression by the small regulatory RNA species miR-23a and miR-23b, where downregulation of TrxR1 induced myogenic differentiation of mouse myoblasts [107]. It was also found that expression of some of the rarer splice variants of TrxR1 inversely correlates with differentiation in squamous carcinoma [108]. Surprisingly, a Pro190Leu mutation in TrxR1, as a result of a homogenous SNP variant that yields lower cellular activity of the enzyme, is linked to generalized epilepsy [109]. Heterozygous SNP variants in the TXNRD2 gene that lead to mutations in the FAD-binding domain of TrxR2 are instead linked to dilated cardiomyopathy [110]. These observations agree rather well with the phenotypes of mouse knockout models, showing that loss of Txnrd1 often promotes increased differentiation while loss of Txnrd2 mainly gives strong effects in heart development or hematopoiesis (see next).

Knockout models

Although whole animal knockout models in mice with deletion of Txnrd1 or Txnrd2 are lethal, conditional knockouts show evident signs of major effects on cell patterning programs. Also analyses of the whole animal knockout of Txnrd1 revealed such effects, with no signs of increased oxidative stress but instead an almost complete lack of mesoderm formation [33]. When Txnrd1 is conditionally deleted in hepatocytes, the mice display larger livers presenting a striking metabolic switch towards glycogen accumulation together with very strong Nrf2 activation [62, 111–113]. Conditional knockout of Txnrd1 in neurons yield severe ataxia and disorganized brain development with cerebral hypoplasia [114]. Whole-animal knockout of Txnrd2 mainly gives lethal deficiencies in heart development and hematopoiesis [115], with major phenotypes also upon conditional knockout in heart [116] or cardiomyocytes [117], but no evident effect when deleted in B- and T-cells [118]. Lack of Txnrd2 in chondrocytes promotes proliferation, differentiation as well as apoptosis [119], while conditional knockout in endothelial cells displays a phenotype only under stressed conditions [120]. An interesting effect was found using Txnrd2 knockout mouse embryonic fibroblasts inoculated as subcutaneous tumors, which impaired the angiogenic switch as compared to controls [121].

In isolated Txnrd1 deficient mouse embryonic fibroblasts there is a major effect on cell differentiation with increased adipogenesis and insulin responsiveness [37]. It should be emphasized that in those TrxR1-deficient cells, Trx1 was still found to be reduced [37], which is likely to be explained by Trx1 reduction through GSH-dependent glutaredoxins [122]. These observations are noteworthy, as they suggest that effects of TrxR1 impairment on cellular phenotypes are not necessarily due to impaired Trx1 functions. Using the Txnrd1 deficient mouse embryonic fibroblasts it was however also shown that TrxR1 is an essential part for cellular antioxidant defense, but only in sparsely cultured cells stressed with high glucose and grown in medium lacking pyruvate or other antioxidants [38]. The same cells furthermore show increased responses to PDGF treatment due to increased inhibition of PTP1B [58], which again illustrates direct links of TrxR1 to cellular signaling pathways.

Molecular mechanisms by which thioredoxin reductases can control cellular patterning programs

With the several observations of disturbed cell patterning upon modulation of TrxR1 or TrxR2 activities, the question is by what molecular mechanisms these effects may arise. Here we shall discuss some of the possible mechanisms that could be involved and give references to key supporting observations in the literature. For a graphical summary of pathways that may be involved see Figure 2.

Figure 2. Cellular targets of mammalian TrxR1 and TrxR2.

This scheme briefly summarizes major downstream targets of the NADPH-dependent cytosolic enzyme TrxR1 (encoded by TXNRD1) and the mitochondrial TrxR2 (encoded by TXNRD2) through the activities of their principal substrates Trx1 and TRP14 (encoded by TXN and TXNDC17) in the cytosol and Trx2 (TXN2) in the mitochondria. The scheme summarizes substrates containing disulfides that are reduced as well as additional activities, as indicated. Note that this is a highly simplified scheme and that many additional targets exist that in turn may affect cell patterning. See text for further details.

Thioredoxins are well known as powerful and versatile protein disulfide reductases. Indeed, most of their actions are likely to be mediated mainly by catalytic reduction of disulfides in target proteins. This holds true for Trx-mediated reactivation of classical Trx substrates such as oxidized ribonucleotide reductase, peroxiredoxins, methionine sulfoxide reductases (where MsrB1 however forms a selenenylsulfide that is reduced by Trx), and many other target proteins for the thioredoxin system [14, 17, 51, 55], as also discussed above. However, additional target motif entities on protein Cys residues that may be reduced by Trx1, Trx2 and TRP14, likely to be involved in modulation of signaling pathways, include S-nitrosylated [12, 57, 123–126] or persulfidated [56, 127] motifs in target proteins. Also, in regulation of the activity of protein-tyrosine phosphatase-1B (PTP1B) the oxidized target seems to be a sulfenylamide motif that can be reduced by either Trx1 or TRP14 [128–130].

The activities of apoptosis signal regulating kinase 1 (ASK1) are also influenced by Trx1, yet by a distinct mechanism. Reduced Trx1 binds to ASK1 in an inhibitory manner, while oxidized Trx1 does not, thereby activating ASK1 under conditions where the active site motif of Trx1 becomes oxidized [16, 53, 131–133]. Trx2 regulates mitochondrial ASK1 in a similar manner [131]. The binding of reduced Trx1 to the Trx-interacting protein (TXNIP) can have a similar function; when Trx1 becomes oxidized it will release TXNIP, with TXNIP subsequently binding to the NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inflammasome that in turn triggers inflammatory signaling cascades [134–136]. Thus, it is not merely through potent disulfide reduction that the Trx system asserts modulatory roles in signaling, but also through more elaborate molecular interactions. The final outcome in signaling and modulation of cell patterning will depend upon a combination of the nature of the posttranslational modification that is targeted, the change in function incurred by the modification, the exact signaling role(s) of the target protein(s), and the overall cellular context in which modulation of such modifications occur. Considering the complexity of the web of signaling networks in a cell, it becomes obvious that although it is clear that the thioredoxin system modulates redox signaling pathways in cells, it seems almost impossible to fully predict the final impact on cellular phenotypes that may be the result of a particular modification in a particular cell. Notwithstanding this fact, we shall next discuss in further details a number of specific signaling systems that may be involved.

S-nitrosylation pathways

Nitrosylation of Cys residues in proteins, a process dependent upon regulated production of the gaseous signaling molecule nitrous oxide (NO) and its derivatives, is becoming recognized as a common mechanism for regulating protein functions in diverse signaling pathways [126, 137–140]. The human Trx1 active site has two Cys residues (Cys32 and Cys35) and in addition Trx1 has three structural Cys residues (Cys 62, Cys69 and Cys73), which can be nitrosylated. Potentially Cys73 of Trx1 might be involved in transnitrosylation reactions [124]. Also Trx2 can participate in transnitrosylation reactions, while during Fas-induced apoptosis, mitochondrial caspase-3 is activated by Trx2 dependent denitrosylation [125, 141, 142]. Trx2-mediated modulation of caspase-3 nitrosylation status can directly regulate the cellular phenotype of microglia [123]. In addition to Trx system-mediated denitrosylation activities, reduced lipoic acid is also able to carry out this reaction [143]. In this context it can be noted that lipoic acid is also a substrate of TrxR [144]. Several studies indicate that the Trx system is likely to modulate the balance between nitrosylation and denitrosylation reactions, and thereby control NO signaling pathways [125, 140, 145]. However, the exact mechanisms by which the Trx system regulates NO signaling is still unclear. Intriguingly, S-nitroso-glutathione (GSNO), which is spontaneously formed upon reaction of NO with GSH is a potent substrate for TrxR, with the reaction resulting in renewed liberation of GSH and NO [146]. GSNO itself was found as a positive regulator of Trx1 activity in rat pulmonary artery smooth muscle cells by lowering the expression levels of the Trx1-inhibitory protein Txnip [147], suggesting a highly intricate network of regulation. It should furthermore be noted that GSNO reductase (GSNOR, also known as class III alcohol dehydrogenase, ADH5) is a GSNO-reducing enzyme using NADH, which acts in parallell with the Trx system and NO synthases for regulation of nitrosylation pathways. In contrast to the Trx system, GSNOR can however not directly reduce nitrosylated proteins; thus it seems likely that the Trx system is the major cellular reducing system for nitrosylated Cys residues (so called SNO moieties) as found in proteins [12, 125, 148].

Protein phosphorylation cascades and PTP activities

Activation of tyrosine kinase phosphorylation cascades induces different cellular programs that control important events in cell fate determination, such as proliferation, migration and differentiation [149]. Protein phosphorylation cascades are mainly counteracted by the superfamily of protein tyrosine phosphatases (PTPs). Their activities are dependent upon conserved active site cysteine residues, which are sensitive to inhibition by oxidation. Inactivation of PTPs by oxidation is an important mechanism that is initiated by the onset of NADPH oxidase activity in response to growth factor receptor activation [150]. Local bursts of H₂O₂ thus become prerequisites for several pathways of protein phosphorylation cascades [151, 152]. Both PTPs and protein tyrosine kinases can be redox regulated and also modulated by the Trx system. Examples of such mechanisms shall here be further discussed.

The onset of lipolysis as stimulated by catecholamines or glucagon involves accumulation of cyclic adenosine monophosphate (cAMP), produced by adenylate cyclase, which initiates activation of cyclic AMP-dependent protein kinase (PKA) and triggers protein phosphorylation cascades [153]. Opposing metabolic signals are mediated through the actions of insulin that stimulate activation of lipogenesis. Interestingly, ligand dependent activation of the insulin receptor stimulates H₂O₂ production, which inactivates PKA by oxidation and formation of an intramolecular disulfide, which in turn is reduced and reactivated by TrxR1 and Trx1 [154]. In this context it is interesting to note that in yeast, the Trx1-dependent Prx1 orthologue Tsa1 is required for the increased lifespan seen upon caloric restriction, through interactions with PKA function [155]. Similar mechanisms of oxidative inactivation also takes occur with protein kinase C (PKC) isoforms, with reduction and reactivation being facilitated by TrxR and Trx [156, 157]. Intriguingly, it has been shown that PKC activity may in turn negatively regulate TrxR expression in human umbilical vein endothelial cells [158]. This might indicate a potential feedback loop of PKC signaling where TrxR and PKC activities counteract and balance each other.

The dual specificity phosphatase and tensin homolog, PTEN, is an important tumor suppressor that regulates cell proliferation, migration and survival. PTEN controls signaling through several growth factor receptors by dephosphorylating phosphatidylinositol 3,4,5-trisphosphate (PIP3), the product of phosphatidyl inositol 3-kinase (PIK). The active site cysteine (Cys124) of PTEN is readily oxidized during cellular responses as triggered by several stimuli, including growth factors, cytokines and hormones [159, 160]. The Trx system has been shown to effectively re-activate oxidized PTEN [129, 160], hence again regulating key intracellular signaling pathways that determine cellular fate.

Intriguingly, differential specificity has been identified for the Trx system with regards to reactivation of different PTPs. PTP1B, in contrast to SHP-2, displays an increase in its oxidation in MEF cells where TrxR1 is deleted [58]. This also correlates with an increased PDGF ligand-dependent phosphorylation of the PDGFRβ receptor site pY579–581, which is regulated by PTP1B. In contrast the phosphorylation site of SHP-2 was unaffected by the cellular TrxR1 status [58]. Differential sensitivity between PTP1B and SHP2 towards the Trx system was also confirmed by in vitro analyses [58, 161]. Recently, it was revealed that TrxR1 is also able to directly reduce a transient oxidation intermediate of PTP1B, potentially its sulfenic acid form [162], thus suggesting multiple modes of regulation of PTP1B by the Trx system. Indeed, TrxR1, Trx1 and interestingly also TRP14 are all potent regulators of PTP1B activity [128, 129, 161, 162]. This agrees well with the finding that the TrxR1 inhibitor auranofin inhibits cellular PTP activity and thereby augments signaling through src tyrosine kinases leading to ERK1/2 phosphorylation [163]. Similarly, TrxR-dependent pathways, but not GSH, can revive the activity of the dual specificity phosphatase Cdc25, which is a PTP that is rapidly oxidized by H₂O₂ [164].

Most growth factor receptor-linked signaling pathways, including those triggered by insulin, EGF, PDGF, FGF, Wnt, TGF or TNF, depend upon protein phosphorylation cascades. This is exemplified by the transmembrane receptor-tyrosine kinases (RTKs) linked to mitogen-activated protein kinase (MAPK) pathways [149, 165]. These pathways are also known to be redox modulated, i.e., altered in their signaling profiles by redox related events [166]. It is thereby not surprising that these pathways are also mediated by alterations in activities in proteins of the Trx system [61, 167]. More specifically, it seems clear that oxidative inactivation of PTPs is an important prerequisite for phosphorylation cascades to occur [130, 151, 168] and in this context the opposing PTP-activating capacity of the Trx system should be similarly important for inhibition of signaling [128, 129].

AMPK and mTOR

Two proteins that play key roles in control of metabolic pathways in response to diverse growth conditions are the AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR). AMPK is activated in response to low ATP levels and helps to control growth, metabolism, autophagy and cell polarity [169]. Partly downstream of AMPK, mTOR is activated by nutrient-rich conditions and can in many aspects antagonize the cellular functions of AMPK [170]. The two pathways present significant crosstalk with each other and also show tissue- and organ-specific regulatory circuits and activities [171]. Interestingly, several activities in these pathways are modulated also by the Trx system. AMPK itself can be inhibited by oxidation through the formation of a disulfide, which is a substrate of Trx1; thereby Trx1 can be considered an essential cofactor for activation of AMPK [172]. It is in this context noteworthy that Akt is strongly upregulated upon Txnrd1 deletion [37], with Akt inhibiting AMPK and activating mTOR [170, 171]. However, in cardiomyocytes Trx1 was found to preserve the activities of mTOR by directly reducing a disulfide formed in mTOR upon oxidative stress [173]. Thus, it seems clear that the Trx system modulates both AMPK and mTOR, while the outcome of such modulation is likely to be both context- and cell type-dependent.

Regulation of transcription factor activities

Most if not all signaling pathways that alter cellular phenotypes converge upon transcription factors that ultimately regulate cell fate through modulated transcriptional programs. Also in this context the Trx system is important, as it directly regulates the activities of several transcription factors. Many transcription factors are redox regulated, including NF-κB, HIF, Nrf2, Oct-4, β-catenin, Notch, and c-Myc, with most of these being important regulators of development or cellular differentiation [4, 174, 175]. Examples of specific transcription factor that are modulated by the TrxR1 status are many, including the observations that TrxR1 is important for maturation of functional p53 [37, 176–178], may act as an important repressor of Nrf2 activation [10], and in the form of a specific splice variant affects estrogen receptor signaling in the nucleus [46]. The TrxR1 substrate TRP14 is a repressor of NFκB signaling [60, 61], and nuclear Trx1 modulates the activities of AP1 through Ref-1 [179], p21 through p53 [180], and Wnt/beta-catenin signaling through the Trx-like protein nucleoredoxin (NXN) [181, 182]. The Trx system thereby carries out specific roles in the nuclei that directly affect transcriptional responses to cellular signaling pathways, which in turn may have been initiated at the cell membrane or in the cytosol in processes that may also be modulated by the thioredoxin system, as discussed above. The complexity by which the Trx system may affect signaling pathways is thus extensive. Here we shall specifically discuss some of the key transcription factors known to be regulated by the Trx system.

Nrf2

Nrf2 is an important cap-n-collar BZip transcription factor that plays a predominant role in cellular stress responses across all metazoan phyla and in different cell types [183–187]. The mechanisms of Nrf2 activation are of specific interest in relation to TrxR1 modulation. During unstressed conditions the Kelch-like ECH-associated protein-1 (Keap1) in the cytosol targets Nrf2 for ubiqutination and rapid proteasomal degradation. Upon oxidative or electrophilic stress, however, Keap1 becomes modified and cannot release Nrf2 for degradation. As a consequence newly synthesized Nrf2 transits to the nucleus, where it heterodimerizes with small BZip Maf proteins. Nrf2/Maf heterodimers subsequently bind so called “antioxidant response elements” in the regulatory regions of genes encoding important proteins of the GSH and Trx systems as well as Phase-II and -III drug-metabolism enzymes [183–187]. Nrf2 also drives metabolic realignments to support these systems, including modulation of energy metabolism with augmented NADPH production [4, 188, 189]. It is in the context of this review article important to note that a wide range of conditions that lower cytosolic TrxR1 activities, including the use of inhibitors of the enzyme or when its activity is lowered by genetic means, typically leads to very strong Nrf2 activation [190].

Some evidence suggests that Nrf2 might also participate in normal developmental processes. For example, in intestinal stem cells of Drosophila melanogaster, constitutive Nrf2 activation sustains quiescence, in part via up-regulation of genes such as GCLC [191]. As an inducer of antioxidant defenses and detoxification, Nrf2 can also protect normal cells from oxidatively induced mutations that may lead to cancer initiation. However, Nrf2 activation in established cancer cells likely promotes cancer progression, by the resultant increased cellular protection against endogenous oxidative stress, cytotoxic immune responses, and therapeutic oxidative (e.g., in radiation therapy) or cytotoxic/electrophilic (e.g., in chemotherapies) challenges [183, 192]. Moreover, Nrf2 activation can accelerate malignant cell growth [193] and Nrf2 is indeed typically activated in many tumors [183, 194] and is critical for malignant progression of pancreatic, breast, and lung cancers [192, 195, 196].

HIF

The HIF1 transcription factor consists of two subunits, HIF1β (ARNT) and HIF1α [197]. During normoxia, a specific proline residue on HIF1α is hydroxylated by prolyl hydroxylase domain proteins (PHD), allowing the recognition and ubiquitination of HIF1α by the Von Hippel-Lindau (pVHL) protein followed by proteasomal degradation. Under hypoxic conditions (O₂ below 3%), PHDs are inactivated by a shift from Fe³⁺ to Fe²⁺ in their active center and as a result, HIF1α becomes stabilized and translocates to the nucleus, where it together with HIF1β induces HIF target genes involved in e.g. the adaptation to hypoxia, angiogenesis, glucose transport, survival and invasion [198]. Interestingly, Trx1 strongly stimulates both the levels and transcriptional activities of HIF1 under normoxic as well as hypoxic conditions [199, 200]. Several pharmacological treatments that target the Trx system or redox states of cells hence affect HIF1 signaling, and there is also a significant crosstalk between HIF1, Nrf2 and NF-κB under such conditions [201].

NFkB

Nuclear transcription factor kappa-B (NF-κB) guides inflammatory pathways and cellular responses to inflammation, with intricate mechanisms in control of its activation [4, 202]. Under basal conditions, NF-κB is kept inactive in the cytosol by binding to I B, the inhibitor of NF-κB. Upon activation, a phosphorylation cascade results in degradation of I B and nuclear translocation of NF-κB [4, 202, 203]. Upon constitutively activated Wnt signaling, Rac1-driven H₂O₂ production by NADPH oxidase activation becomes required for NF-κB activation with initiation of colon tumorigenesis [204]. In the context of the Trx system, the alternative TrxR1 substrate TRP14 was initially discovered as a protein repressing NF-κB signaling through reduction of LC8 cytoplasmic dynein light chain [61]. Also Trx1 counteracts NF-κB but likely by other mechanisms than TRP14 not involving LC8 [205]. TrxR1, but not TrxR2, was shown to facilitate NF-κB signaling [206], but as those experiments were based upon forced overexpression of the selenoprotein it is also possible that formation of SecTRAPs contributed to the observed effects (see discussion above).

p53

The important tumor supressor p53 has long been known to be a redox sensitive transcription factor [207]. It was early shown that Trx1 augments p21 transactivation by p53, at the same time as Trx1 becomes targeted to the nucleus [180]. It was also shown in several studies that an intact TrxR1 activity is required for maturation and support of transcriptional activity of p53 [177, 178, 208, 209]. Since physiological functions of p53 include regulation of cell patterning during development and control of stem cell differentiation [210], it seems clear that effects on cell patterning upon Trx system aberrations may in part be due to modulated p53 function.

STAT3

STAT3 is a protein that mediates the expression of a variety of genes in response to cell stimuli and plays key roles in many cellular processes in cell patterning, moreover linked to NF signaling pathways [211]. With regards to STAT3 signaling in relation to the Trx system it is thus interesting to note that STAT3 activity upregulates TRP14 transcription, encoded by the TXNDC17 gene [212], and that thioredoxin interacting protein (TXNIP), an endogenous Trx1 inhibitor [213–215], can inactivate STAT3 [216]. The STAT3 regulation by H₂O₂ and its modulation by the Trx system, through several direct and indirect mechanisms via Prx2, Trx1, TRP14 and TXNIP [212, 216, 217], is another illustration of the intricate webs of highly complex signaling pathways that link the Trx system to cellular patterning programs.

Conclusions

Here we have discussed several key studies that indicate that the Trx system has important roles in control of cell patterning, in addition to its more known functions in support of reductive enzymes and antioxidant defense. We have focused upon studies showing effects on cell patterning by genetic manipulation of TrxR1 or TrxR2 expression in diverse model systems, and we have discussed the downstream effects and molecular mechanisms that may explain the observed phenotypes. It is safe to conclude that much of the details in these mechanisms are yet insufficiently understood, but it is just as evident that the Trx system should be considered as an important player in control of cell patterning processes.

Highlights.

• The thioredoxin system is important for support of cellular reduction pathways and antioxidant defense

• The thioredoxin system is also crucial in redox control of cellular signaling pathways

• The major phenotypes triggered by aberrations in the thioredoxin system are altered cell patterning programs

• This review discusses the possible molecular mechanisms underlying these observations