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Emerging Evidence of the Significance of Thioredoxin-1 in Hematopoietic Stem Cell Aging

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The United States is undergoing a demographic shift towards an older population with profound economic, social, and healthcare implications. The number of Americans aged 65 and older will reach 80 million by 2040. The shift will be even more dramatic in the extremes of age, with a projected 400% increase in the population over 85 years old in the next two decades. Understanding the molecular and cellular mechanisms of ageing is crucial to reduce ageing-associated disease and to improve the quality of life for the elderly. In this review, we summarized the changes associated with the ageing of hematopoietic stem cells (HSCs) and what is known about some of the key underlying cellular and molecular pathways. We focus here on the effects of reactive oxygen species and the thioredoxin redox homeostasis system on ageing biology in HSCs and the HSC microenvironment. We present additional data from our lab demonstrating the key role of thioredoxin-1 in regulating HSC ageing.
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Citation: Jabbar, S.; Mathews, P.;
Kang, Y. Emerging Evidence of the
Significance of Thioredoxin-1 in
Hematopoietic Stem Cell Aging.
Antioxidants 2022,11, 1291. https://
doi.org/10.3390/antiox11071291
Academic Editors: Edward E.
Schmidt, Hozumi Motohashi
and Anna Kipp
Received: 1 June 2022
Accepted: 28 June 2022
Published: 29 June 2022
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antioxidants
Review
Emerging Evidence of the Significance of Thioredoxin-1 in
Hematopoietic Stem Cell Aging
Shaima Jabbar, Parker Mathews and Yubin Kang *
Division of Hematologic Malignancies and Cellular Therapy, Department of Medicine, School of Medicine,
Duke University Medical Center, Durham, NC 27710, USA; shaima.jabbar@duke.edu (S.J.);
parker.mathews@duke.edu (P.M.)
*Correspondence: yubin.kang@duke.edu; Tel.: +1-919-668-2331; Fax: +1-919-613-1319
Abstract:
The United States is undergoing a demographic shift towards an older population with
profound economic, social, and healthcare implications. The number of Americans aged 65 and older
will reach 80 million by 2040. The shift will be even more dramatic in the extremes of age, with a
projected 400% increase in the population over 85 years old in the next two decades. Understanding
the molecular and cellular mechanisms of ageing is crucial to reduce ageing-associated disease and to
improve the quality of life for the elderly. In this review, we summarized the changes associated with
the ageing of hematopoietic stem cells (HSCs) and what is known about some of the key underlying
cellular and molecular pathways. We focus here on the effects of reactive oxygen species and the
thioredoxin redox homeostasis system on ageing biology in HSCs and the HSC microenvironment.
We present additional data from our lab demonstrating the key role of thioredoxin-1 in regulating
HSC ageing.
Keywords: hematopoietic stem cells; ageing; thioredoxin; redox systems
1. Introduction
Ageing is a natural process in all living organisms and represents a progressive decline
in functional activities. Ageing is associated with many pathophysiological disorders
including autoimmune diseases, cancers, diabetes, cardiovascular diseases, and neurode-
generative conditions [
1
]. Hematopoietic stem cells (HSCs) are a rare population of cells
that reside in specialized bone marrow (BM) niche and are characterized by their ability
to self-renew and differentiate into hematopoietic multipotent progenitors (MPPs) and
hematopoietic progenitor cells (HPCs) to maintain hematopoiesis and an immune system
during the entire lifespan of the organism [
2
]. HSCs will age as do all other cells. Dur-
ing ageing, the functionality and integrity of HSCs decline, and various factors such as
increased cellular metabolic demands, an altered BM microenvironment, DNA damage,
exposure to high levels of free radicals, and epigenetic changes can all contribute to shift
HSCs towards ageing phenotypes. The ageing of HSCs is the key process underlying
the decline in immune function (so called “immunosenescence”), the lineage skewing of
hematopoiesis, and the increase in the incidence of hematological malignancies seen in the
elderly population [
3
] (Figure 1). Currently, the molecular and cellular pathways driving
the ageing of HSCs are not well characterized. The continuous existence of this gap in
our knowledge poses a significant obstacle to our efforts to attenuate ageing-associated
disease and to improve the quality of life for the elderly. In this review, we summarized
the biological concepts of HSC ageing including the hallmarks of HSC ageing and the
molecular mechanisms that drive HSC ageing. We reviewed the effects of reactive oxygen
species (ROS) on HSC ageing, with a particular emphasis on the roles of thioredoxin-1 in
HSC ageing. Understanding the mechanisms of HSC ageing and the effects of thioredoxin-1
provides a foundation for us to develop novel approaches to rejuvenate aged HSCs and
Antioxidants 2022,11, 1291. https://doi.org/10.3390/antiox11071291 https://www.mdpi.com/journal/antioxidants
Antioxidants 2022,11, 1291 2 of 22
to attenuate tumorigenesis and thus has important implications in the current era, where
there is a critical demographic shift towards an ageing population in the USA.
1
Figure 1.
Ageing-associated changes in HSCs. Young HSCs exhibit high self-renewal and recon-
stitutional capacities, a high homing frequency, low ROS, and a balanced lineage output. Aged
HSCs are characterized by a decline in self-renewal and reconstitutional capacities, high ROS, a low
homing frequency, and increased myeloid lineage skewing. Created with BioRender.com, accessed
on 19 June 2022.
Antioxidants 2022,11, 1291 3 of 22
2. Background: Biological Concepts of HSC Ageing
HSCs can both self-renew and differentiate to continuously replenish all lineages of
blood cells throughout life. In the 1970s and 1980s, scientists postulated that HSCs are
ageing-exempt due to their telomerase activity, which might preserve HSC longevity at
least beyond the expected human lifespan [
4
,
5
]. Our understanding changed when Sean
et al. made their landmark discovery, showing that HSCs isolated from older mice exhibited
a reduced ability to engraft lethally irradiated mice compared to young HSCs. Although
the mechanism behind this biological phenomena was not addressed [
6
], this seminal
observation laid the foundation for our current research addressing the differences between
juvenile and aged HSCs.
The ageing of HSCs is complex, and there is no standardized model defining the key
mechanisms of HSC ageing. Furthermore, there is no single cell surface marker that can be
used reliably to sort and characterize aged HSCs, nor a golden index for the measurement
of HSC kinetics and cell cycle dynamics to define the biological age of HSCs. Most of the
studies in the field of HSC ageing research have been conducted by enriching HSCs from
aged mice and comparing their biological functions to those isolated from young mice.
The hallmark of HSC ageing is their reduced ability for self-renewal and reconstitution
potency following serial transplantations in myeloablative recipient mice. Although the
number of mouse HSCs increases with age, the ability of aged HSCs to self-renew and
reconstitute erythroid and lymphoid lineages in serial transplant recipient mice is impaired.
Aged HSCs lose their original compartment signature, and their multilineage differentia-
tion features are reduced due to stress and exhaustion [
7
10
]. Aged HSCs show lineage
skewing with more myeloid differentiation and less lymphoid cell production. Using an
inducible Fgd5-based HSC lineage tracing model, scientists have found that HSCs usually
preferably differentiate to the myeloerythriod lineage and less to the lymphoid (adaptive
immunity) lineage. In the setting of acute thrombocytopenia, HSCs rapidly differentiate
into the platelet lineage [
8
,
10
13
]. The differentiation kinetic of aged HSCs seems to be
defective. In fact, in old mice and humans, HSCs increase their proliferation and expansion
frequency and skew their differentiation to the myeloid lineage (and in some cases, myeloid
malignancies) and less to the erythroid and lymphoid cells [
14
16
]. The lineage skewing of
aged HSCs is consistent with the clinical observation that hematology disorders such as
anemia and hematological malignancies such as leukemia, myeloma, and lymphoma are
linearly correlated with lifespan [1721].
Aged HSCs also exhibit defects in stem cell homing and engraftment. It was reported
that the homing efficiency of aged HSCs was significantly reduced compared to young
HSCs. Liang and his colleague transplanted bone marrow (BM) cells from young or old
C57BL/6 mice into old or young Ly5 congenic mice. Interestingly, they found the homing
efficiency of HSCs from the old mice (2 years old of age) to be reduced by more than
threefold compared to that of HSCs from young mice (2 months of age) [
22
]. Most of the
studies examining the homing efficiency of HSCs involve transplanting BM HSCs into
lethally irradiated hosts. These assays test not only the role of HSCs themselves but also
the impact of microenvironmental factors on the seeding frequency of HSCs. To dissect
out the effect of HSCs themselves versus the impact of the microenvironment, Dykstra and
his colleague developed a barcode tool and conducted a co-homing study by labeling old
and young BM HSCs with GFP at a fixed ratio and transplanting the HSCs into lethally
irradiated or non-irradiated old mice. Their results revealed that the homing frequency of
BM HSCs improved in non-irradiated old recipient mice and that old/young HSCs that
successfully homed into old recipient mice lost their compartment integrity compared to
the pre-homed HSCs [23]. Figure 2summarizes some of the hallmarks of HSC ageing.
The cellular and molecular pathways driving HSC ageing are complex and remain
an area of active investigation. HSCs reside in the BM matrix and are surrounded by
non-hematopoietic cells. Factors that are related to HSC ageing are typically classified as
intrinsic and extrinsic (microenvironmental) [24].
Antioxidants 2022,11, 1291 4 of 22
2
Figure 2.
Hallmarks of HSC ageing. Aged HSCs exhibit impaired self-renewal and reconstitutional
capacities and increased replicative stress. They are also characterized by cell senescence, increased
cell apoptosis, and declined mitochondrial biological functions. Aged HSCs are associated with a
deficiency in the DNA repair pathway, higher mutation rates, increased myeloid lineage skewing,
and an increased incidence of hematological malignancies. Created with BioRender.com, accessed on
19 June 2022.
2.1. Intrinsic Factors Driving HSCs Ageing
There are several intrinsic mechanisms that have previously been identified as con-
tributing to ageing-related changes of HSCs. Although much of this discussion will address
these mechanisms separately, the intrinsic factors that contribute to HSC ageing are highly
interconnected and interdependent.
2.1.1. DNA Damage Response and HSC Ageing
DNA damage response (DDR) is a cellular intrinsic factor that shifts HSCs towards
ageing phenotypes and can be triggered by physical (low dose radiation), chemical (geno-
toxic agents), or biological insults (replication stress or oxidative stress). During cell cycle
arrest, the DNA proofreading enzymes are activated, and DNA double stranded breaks
are repaired either by homologous recombination (HR) or by non-homologous end joining
(NHEJ) [
25
]. NHEJ is thought to be error prone and can lead to DNA mutations, while HR
is considered as a DNA repair error-free pathway. With each cell division and expansion,
there are increased risks for replication stress and cellular proofreading machinery errors,
both of which cause DNA damage. Typically, most HSCs reside in a relatively hypoxic niche
and are characterized by low metabolic activity and a lower proliferation rate, which make
HSCs less likely subject to NHEJ repair [
25
,
26
]. However, HSCs residing in the G0 cell cycle
are more vulnerable to the NHEJ repair process, resulting in the loss of DNA integrity [
26
].
Additional lines of evidence suggested that the upregulation of p21 in HSCs in response
to DNA damage promotes this faulty DNA repairing system, which may ultimately lead
HSCs towards exhaustion and aged phenotypes [27].
DNA damage repair mechanisms are related to HSC stemness and differentiation.
Through the tumor suppressor p53 pathway, DDR activity can serve as a signal for HSCs to
Antioxidants 2022,11, 1291 5 of 22
switch from self-renewal to a commitment to differentiate into progenitor cells [
28
]. p53, as
the “cellular gatekeeper”, is essential in coordinating the cellular responses to a broad range
of cellular stress factors [
29
]. Studies suggested that p53 plays an important role in the
regulation of HSC quiescence and senescence [
27
,
28
], with declining p53 function associated
with longevity in naturally aged mice [
27
,
30
]. DDR is a key component regulating the long-
term self-renewal integrity and differentiation efficiency of HSCs, and the accumulation of
DDR through the lifespan can shift HSCs towards ageing phenotypes.
2.1.2. Senescence and HSC Ageing
In HSCs, cell senescence is characterized by terminal cell cycle arrest in which the
cells are unable to undergo self-renewal or differentiation. The clearance of senescent cells
using progeroid transgenic mice successfully delayed the progression of ageing-associated
disorders [
31
,
32
], suggesting that cell senescence plays a major and likely causative role in
HSC ageing-related phenotypes. The cell cycle-dependent kinase inhibitors p16
INK4a
and
p14(ARF) are important molecules regulating mammalian cell senescence [
33
,
34
]. In old
murine HSCs, the upregulation of Notch signaling through the activation of the TGF-
β
/
pSmad3 pathway, which led to the inhibition of p16
INK4a
, significantly improved the
regenerative capacity of aged HSCs [
31
36
]. The administration of fibroblast growth
factor 7
in murine models resulted in the repression of p16
INK4a
and the partial rejuvenation of
early T cell progenitors [
34
]. The overexpression of p16
INK4a
in peripheral blood immune
cells is positively correlated with human chronological age, suggesting that p16
INK4a
is
a potential biomarker of human molecular age [
34
]. However, there are also studies that
suggested a more limited role of p16
INK4a
in the ageing-related changes of HSCs and
that the decline of HSC functions during ageing is not dependent on the induction of p16
INK4a
but is instead mediated by other, currently undefined mechanisms [
37
39
]. Protein
polybromo-1 (PB1), also known as BRG1-associated factor 180 (BAF180), plays a protective
role in HSC cell senescence through counteracting the p21 transcription factor. The deletion
of BAF180 in murine HSCs exhibited deleterious effects on the long-term regeneration and
differentiation potency of HSCs [40].
Other intrinsic pathways involved in HSC senescence pathways include p16, JAK/STAT,
NF-
κ
B, the mammalian target of rapamycin (mTOR) pathways, the TGF-
β
signaling path-
way, the Wnt pathway, and reactive oxygen species (ROS), among others [
24
] (Table 1).
Targeting cell cycle-dependent kinase inhibitors (CKI) such as p21, mTOR, and p38 mitogen-
activated protein kinase (MAPK) was reportedly able to successfully counteract cell cycle
defects in order to prevent HSC exhaustion and senescence and to rejuvenate hematopoiesis
in elderly mice [4143].
Table 1. Selected molecules and pathways in the regulation of HSC senescence.
Cell Senescence Molecules and Pathways Functional Activities References
p53-p21 axis
Telomerase activity
Oxidative stress
Terminal cell cycling arrest
[44]
EZh1 and EZh2, also known as
polycomb protein members
Differentiation
Self-renewal
Genomic integrity
[45]
SA-β-galactosidase (SA-β-Gal) and lipofuscin
Clonogenic capacity
Oxidative stress
DNA damage
[46]
Bmi1, a member of the Polycomb group proteins Reconstitution, repopulation, and self-renewal capacities
Mitochondrial production of ROS [47,48]
Ink4a/Arf transcription factors
Apoptosis
Cell cycle arrest
Senescence-associated heterochromatic foci (SAHF)
[49,50]
Antioxidants 2022,11, 1291 6 of 22
Table 1. Cont.
Cell Senescence Molecules and Pathways Functional Activities References
Arf/P53 pathway
HSC expansion and self-renewal
Apoptosis
Exhaustion and stress proliferation response
[5153]
p38/MAP kinase signaling pathway
DNA damage
Oxidative stress
Telomerase activity
Exhaustion and stressful replication
[5456]
Ataxia-telangiectasia mutated (ATM) and
Telomerase reverse transcriptase (TERT)
Oxidative stress
Biological functions
Self-renewal capacity
Apoptosis
[57]
Chemokines and cytokines including IL-8
(CXCL8), GROα(CXCL1), GROβ(CXCL2),
GROγ(CXCL3), MCP-1 (CCL2), MCP-2
(CCL8), MCP-4 (CCL13), MIP-1α(CCL3),
MIP-3α(CCL20), and HCC-4 (CCL16).
Seeding efficiency and homing
Differentiation
Mobilization and migration
Proliferation and expansion
[5861]
2.1.3. Epigenetic Regulation and HSC Ageing
The loss of epigenetic regulation (such as DNA methylation, histone modifications,
and noncoding RNA) is also involved in HSC ageing. Amplification in the methylation peak
at the promotor region of genes associated with HSC self-renewal and differentiation capac-
ity contributes to HSC biological dysfunctions [
14
,
62
]. DNA methyltransferase enzymes
(Dnmt1, Dnmt3a, and Dnmt3b) are responsible for de novo DNA methylation and for main-
taining and regulating DNA methylation islands throughout the lifespan [
62
65
]. Dntm1
plays a coordinating role to balance between self-renewal and lineage differentiation output
in HSCs [
63
]. The deletion of Dnmt1 resulted in a dramatic reduction in HSC self-renewal
and skewed HSC differentiation towards myelopoiesis [
63
65
]. HSCs with Dntm3a
/
and Dntm3b
/
exhibited normal cell expansion and proliferation but showed noticeable
deficits in reconstitutional and differentiation capacities in serial transplantation [
63
,
65
,
66
].
Histone posttranslational modifications are crucial in regulating the access of genes
that encode proteins to determine HSC fate. Emerging evidence shows that open chro-
matic mark H3K4me2 is highly expressed in committed and differentiated HSCs, while
it is inhibited and downregulated in long-term (LT) HSCs and short-term (ST) HSCs [
67
].
Transcriptomic and epigenetic analyses have identified a group of chromatin remodeling
genes including Kdm3a–b, Kdm5b–d, Jarid1b, and Kdm6a–b) that are age-regulated in
HSCs [6870]
. For example, it was recently shown that Kdm5b or Jarid1b are involved in
regulating the expression of genes such as Hoxa7, Hoxa9, Hoxa10, Hes1, and Gata2 that
preserve the self-renewal and proliferation capacities of HSCs [
62
,
71
,
72
]. More evidence
has shown that Jarid1b activity declined in aged murine HSCs, and the downregula-
tion of Jarid1b is strongly associated with the upregulation of cell fate–associated genes
upon lineage commitment [73]. Additional studies are needed to gain a more comprehen-
sive understanding of the role of chromatin modifications and epigenetic regulations in
HSC biology.
2.1.4. Mitochondria and HSC Ageing
Another key factor in HSC ageing that remains incompletely characterized is the
ageing phenotype at the cellular organelle level [
74
]. Mitochondria are the major cellu-
lar organelles responsible for generating higher levels of energy through the TCA cycle
and oxidative phosphorylation [
75
,
76
]. Fetal and neonatal HSCs exhibited higher mito-
chondrial membrane potential (
∆Ψ
mt) and increased mitochondrial dynamics and net
mitochondrial mass compared to aged HSCs [
77
80
]. ROS and stress responses activate the
mammalian sirtuin family SIRT3. SIRT3, also known as the mitochondrial stress regulator
Antioxidants 2022,11, 1291 7 of 22
gene, triggers mitochondrial protein acetylation, increases the expression of mitochondrial
antioxidant enzymes, and enhances ROS scavenging. Recent studies have found that SIRT3
is downregulated in old murine HSCs, which leads to the accumulation of ROS and a
decline in mitochondrial plasticity, with a resultant impairment of HSC reconstitutional
potency. These findings suggest that SIRT3 is a possible cause of ageing-associated changes
in HSCs [81,82].
2.2. Extrinsic Factors Driving HSC Ageing
In addition to intrinsic factors, extrinsic factors in the BM microenvironment niche
could also affect HSC ageing. These extrinsic factors include cellular components of BM
niches, such as hematopoietic cells (megakaryocytes, regulatory T cells, and macrophages)
and non-hematopoietic cells (mesenchymal stromal cells, endothelial cells, perivascular
cells, and nerve fibers), as well as non-cellular components such as growth factors, inflam-
matory cytokines and chemokines, and other soluble factors [
74
,
83
,
84
]. These components
create an HSC extracellular matrix (ECM) and provide support and crosstalk with HSCs.
Any type of pathological or physiological change in the BM microenvironment may impair
the steady state of HSCs. For example, injury or stress on BM microenvironmental cells
activates the local inflammatory response, resulting in changes in the composition and
concentration of local cytokines and interleukins, with important consequences to the
biological functions of HSCs. He et al. showed that tumor necrosis factor alpha (TNF
α
) was
sufficient to activate the ERK-ETS1-IL27R
α
pathway and push HSCs towards replicative
stress and myeloid-biased differentiation [
83
]. Interferon gamma (INF-
γ
), macrophage
colony-stimulating factor (M-CSF), and gram-negative bacterial component lipopolysaccha-
ride (LPS) are able to interact with HSC innate immune receptors and upregulate several
signaling pathways related to HSCs’ biological functional states [
85
87
]. Using single
cell RNA sequencing and genome-wide transcriptomic analysis, scientists identified BM
niche adhesion factors and stem cell factors such as Ang-1, TPO, Wnt, NOTCH, OPN,
and chemokine stromal cell-derived factor-1
α
(SDF-1
α
) as factors that directly regulate
the steady and differentiation states of HSCs [
88
]. Tumor growth factor (TGF-
β
) and
Interleukin 6
(
IL-6
) are overexpressed by aged BM stroma and potentially influence the
expression of genes that are associated with HSC ageing. The downregulation of TGF-
β
and IL-6 had a restorative effect on aged HSCs [
89
]. Plasma cells are particularly enriched in
BM stroma as mice age and express genes associated with inflammatory cytokine response,
which is thought to shift HSCs towards ageing myelopoiesis [
90
]. BM mesenchymal cells
from aged mice have a diminished level of insulin-like growth factor 1 (IGF-1). IGF-1
supports HSC survival, and the overproduction with the IGF-1 ligand was able to restore
the reconstitution capacity of aged HSCs and mitigate the functional myeloid lineage bias
of aged HSCs [91].
BM vascular and endothelial cells provide an important niche for HSCs. Recent studies
demonstrated important roles of several BM endothelial transcription factors (EPCR/PAR1
signaling, Cdc42,Ccr9,Gnrh2, and Lep) in facilitating the retention and repopulation capacity
of HSCs and in reducing the oxidative damage response [
92
]. CD44 is a member of cell
adhesion molecule families expressed by BM niche cells and is crucial in regulating the
migration, homing, and survival of HSCs [
93
]. The deletion of CD44 in neonatal BM
ameliorates the engraftment and homing of HSCs [
93
]. Klf5, a member of the Kruppel-like
family which is mostly expressed by BM epithelial cells, plays a pivotal role in enhancing the
homing, retention, and lodging of BM HSCs. Klf5 was found to correlate with age-related
changes in HSCs [9496].
In summary, the ageing of HSCs is associated with a complex phenotype with char-
acteristic changes from the organelle level to the BM microenvironment. There is a great
clinical significance of these changes, as the impairment of HSC functionality such as the
loss of cell quiescence is one of the hallmarks of the increased incidence of myeloid and lym-
phoid leukemias as well as other hematological malignancies with ageing. The impairment
of immune function (immunosenescence) contributes to an increased susceptibility to infec-
Antioxidants 2022,11, 1291 8 of 22
tion and an increased incidence of autoimmune disease. The myeloid skewing has been
associated with spontaneously increased levels of proinflammatory cytokines/chemokines
and may contribute to frailty and other chronic disease processes in the elderly.
3. Reactive Oxygen Species and HSC Ageing
Reactive oxygen species (ROS), such as superoxide anion (O
2•−
), hydrogen peroxide
(H
2
O
2
), and hydroxyl radical (HO
), are the intermediate products of the cellular respi-
ration process which is necessary for metabolic survival in all aerobic organisms. ROS
are generated during mitochondrial oxidative phosphorylation (OXPHOS) or during the
cellular response to oxidative stress in states such as infection or inflammation. ROS can
be thought of as a double-edged sword: a low cellular ROS level plays a critical role in
promoting cellular biological functions such as cell proliferation and cell survival [
97
100
].
ROS also serve as a critical signaling molecule regulating the mitogen-activated protein
kinase (MAPK) pathway, the phosphoinositide 3-kinase (PI3K) pathway, the Nrf2 and Ref1-
mediated redox cellular signaling pathway, and the Shc adaptor protein family, among
others [
101
]. An increased ROS level due to the failure of the cellular antioxidant system
to scavenge ROS or the overproduction of ROS shifts the cell to a status that is chemically
known as oxidative stress. Oxidative stress in the cytosol or in the mitochondria can have
deleterious effects on the cell biological systems [
102
], causing damage to cellular proteins,
nucleic acids, lipid membranes, and organelles (in particular, the mitochondria).
3.1. Metabolic Status and ROS Production in HSCs
It is well established that oxidative stress has been invariably linked to HSC
ageing [103105]
.
At baseline, HSCs are quiescent and are predominately located in the BM niche with low
oxygen tension (physiologic local hypoxia). The quiescent environment of HSCs is further
supported by BM osteoblasts that also require low oxygen levels and provide HSCs long-
term protection from ROS-related oxidative stress [
79
]. HSCs transition through different
microenvironmental niches during normal physiologic conditions, and, in certain states of
hematologic stress such as hemorrhage, viral infection, and radiation injury, HSCs mobilize
and migrate from the hypoxic environment to the BM blood sinusoidal system with a
highly oxygenic vasculature to increase their catalytic pathways and undergo highly active
proliferation and differentiation to restore hematopoietic homeostasis [106108].
When HSCs divide or expand, there is an increased production of ROS due to the
change in metabolism. HSCs residing in low oxygen environments mostly generate their
fuel through anaerobic glycolysis mediated by pyruvate dehydrogenase kinase 1 (PDK1)
and suppress the influx of glycolytic metabolites into mitochondrial membranes. The
increased anaerobic glycolysis and suppressed cellular respiration results in the low pro-
duction of ROS, which promotes cell quiescence, cell renewal, and survival [
109
,
110
]. On
the other hand, when HSCs are committed to lymphoid and erythroid lineage differentia-
tion, HSCs increase their metabolic flux through pyruvate cycling to significantly increase
ATP production, which in turn results in higher levels of ROS [
86
,
88
]. This may serve as a
feedback loop in which ROS signal a switch of HSC metabolic pathways towards oxidative
phosphorylation, with downstream implications for HSC exhaustion and HSC ageing.
Recently, it has been reported that dormant cells such as HSCs generate ROS through
the phagocyte NADPH oxidase (NOXs) enzymatic system [
111
113
]. ROS produced
by NOXs regulate and contribute to a variety of HSC biological functions. NOX1, 2,
and 4 are expressed in human and murine HSCs [
114
,
115
]. Interestingly, NOX1 and
NOX2 are overexpressed, while NOX4 is downregulated in committed and differentiating
HSCs [113,116,117]
. More importantly, it has been shown that aged murine HSCs exhib-
ited a global increase in ROS, and this is positively associated with the upregulation of
NOX4 [115].
Antioxidants 2022,11, 1291 9 of 22
3.2. Responses to Oxidative Injury
HSCs have various safeguard mechanisms against the cytotoxic accumulation of ROS.
Ataxia telangiectasis mutated (ATM) functions as a sensor of the level of oxidative stress
and phosphorylates several key DNA damage response molecules to protect HSCs from
oxidative DNA damage. Ito et al. found that the ATM regulation of oxidative stress is
crucial for the reconstitutional capacity of HSCs [
118
]. Atm
/
mice older than 24 weeks
had elevated ROS levels and showed progressive bone marrow failure due to defects in
HSC function. Treatment with anti-oxidant agents restored the reconstitutional capacity of
Atm/HSCs [118].
The mammalian sirtuin family members (SIRTs) play important roles in the regulation
of oxidative stress response and have age-specific effects on HSCs. For example, SIRT7
/
mice showed a greater than 40% reduction in the erythroid and myeloid progenies. Fur-
thermore, SIRT7
/
HSCs displayed a dramatic decline in the long-term reconstitution
potency [
80
,
119
]. Aged HSCs exhibited SIRT7 downregulation compared to neonatal and
young HSCs. The deletion of SIRT6 promotes HSC stress replication through the aberrant
activation of the Wnt signaling pathway. The reintroduction of SIRT3 into aged murine
HSCs improves their mitochondrial plasticity and regenerative capacity [80,82,119,120].
PI3K-AKT-mTOR-reactive oxygen species-p53, MDM2, ATM, MAP K, p38, p16, and
p21 have all been identified as oxidative/antioxidant regulatory genes [
121
]. Responding
to oxidative injury involves a complex process characterized by the dephosphorylation of
p53/MDM2 and the activation of the p53 apoptotic signaling pathway [
121
,
122
]. Forkhead
box class O family member proteins (FoxOs) including FOXO1, FOXO3a, and FOXO4 are
essential transcription factors that enhance a return to quiescence and the survival of murine
and human HSCs in response to oxidative stress and damage [
123
]. Free radicles and
oxidative stress cause the phosphorylation of FOXO1, FOXO3a, and FOXO4, which then
activates a group of genes involved in programmed cell death and cell cycle regulation [
123
].
FoxO triple negative mice exhibited a severe decline in HSC integrity and increased myeloid
differentiation bias. In this case, administration of N-acetyl cysteine (NAC) could only
partially rescue HSC defects, which suggested that ROS might not be the main cause
of HSC deficiency seen in FoxO triple negative mice [
123
]. Hypoxia-inducible factor-
1
α
(HIF-1
α
) shifts cellular metabolism from mitochondrial respiration to glycolysis and
reduces ROS production. Cellular anti-oxidant systems such as the superoxide dismutase,
glutathione system, and thioredoxin system can scavenge ROS and reduce the level of ROS.
Mitochondrial superoxide dismutase (SOD2) or cytosolic SOD1 converts superoxide radical
anion (O
2•−
) to hydrogen peroxide (H
2
O
2
), and Glutathione-dependent enzymes catalyze
biological redox reactions and assist in the protection against ROS and oxidative damage.
3.3. Oxidative Injury and HSC Ageing
The imbalance between ROS production and the capacity of anti-oxidant systems to
counteract ROS results in oxidative stress. Oxidative stress induces DNA base damage
and causes the release of free bases and the generation of a basic site, leading to increased
cell cycling and apoptosis as well as compromised self-renewal and the differentiation
of HSCs [
43
,
118
,
124
]. HSCs in the hypoxic BM niche re-localized HIF-1
α
to the nucleus
to promote a plethora of genes that are involved in maintaining the HSC quiescent cell
cycle, cell survival, self-renewal, and appropriate mitochondrial mass. Increases in the
ROS level in HSCs induces the degradation of the HIF-1
α
protein and activates a group
of genes that are associated with cell commitment and differentiation [
125
,
126
]. Nuclear
factor erythroid 2–related factor 2 (Nrf2) has emerged as a master transcription factor that
regulates a plethora of antioxidant genes in HSCs. The conditional deletion of Nrf2 in
mouse models increases HSC sensitivity to ROS and impairs the regenerative capacity of
HSCs, which could not be rescued by the administration of NAC [
127
]. Oxidative stress
may directly trigger a number of stress response signaling pathways such as p38 MAPK
kinase and limit the lifespan of HSCs. The p38 MAPK signaling pathway is involved in
the activation of cellular apoptosis, autophagy, and cellular differentiation [
43
]. In Atm
/
Antioxidants 2022,11, 1291 10 of 22
mice, increased ROS induces the HSC-specific phosphorylation of p38 MAPK and defects
in the maintenance of HSC quiescence. Prolonged treatment with an antioxidant or an
inhibitor of p38 MAPK extended the lifespan of HSCs from wild-type mice in serial trans-
plantation experiments [
43
]. Several studies have found that the thioredoxin interacting
protein TXNIP/p53 axis plays a role in rescuing and reconstituting exhausted HSCs under
oxidative stress [
128
]. Exposing HSCs to oxidative damage or high metabolic rate demands
disturbs the intermolecular disulfide interaction between TXN1-TXIP, resulting in TXN1
nuclear translocation to regulate cell survival and growth through the ERK1/2 MAPK
signaling pathway [
128
130
]. The TXNIP-p38 axis was found to be a promising target in
rejuvenating aged murine HSCs [
131
,
132
]. ROS can also limit the ability of bone marrow
stromal cells to support hematopoietic reconstitution [133].
In summary, ROS concentrations can be thought of as a sensor of the steady state
and stemness capacity of HSCs in various signaling pathways, and the accumulation of
oxidative stress damage may trigger several cell events including apoptosis, autophagy,
and differentiation, which are associated with HSC ageing.
4. The Utility of Antioxidants in the Biology of HSC Ageing
Antioxidants are molecules or proteins with an ability to modulate cellular oxidation
reactions by donating an electron to reduce or prevent the oxidation of oxidizable molecules.
Endogenous antioxidants are categorized into non-protein antioxidant molecules and
antioxidant proteins. Non-protein antioxidant molecules are glutathione, alpha-lipoic acid
(LA), coenzyme Q (CoQ), ferritin, uric acid, and bilirubin. Key protein antioxidants include
superoxide dismutase (SOD), catalase (CAT), thioredoxin, and glutathione peroxidase.
Endogenous antioxidants are the first line of the cellular defense mechanism against
oxidative damages [
134
136
]. The thioredoxin and glutathione systems are the two major
mammalian endogenous antioxidant systems. The thioredoxin and glutathione systems
work in parallel to reduce oxidative metabolites and to supply a steady state of reduction
power in the cytosolic environment to protect cells from the exposure to harmful levels
of ROS [
137
,
138
]. There are a variety of exogenous antioxidant agents such as phenolic
compounds, ascorbic acid, tocopherol, N-acetyl cysteine, and curcumin. Pharmaceutical
companies have developed and are further developing drugs to work as antioxidants or
synergize with antioxidant systems to prevent/delay the ageing process or to rejuvenate
aged cells [138140].
Although oxidative stress and oxidative damage have been established to be highly
associated with the ageing process in a wide variety of organisms, the causative relationship
between oxidative stress and ageing remains unclear. In cell culture systems and in some
animal models, the administration of antioxidants has been shown to rejuvenate aged
HSCs and extend the lifespan of HSCs [
43
,
118
,
141
]. The protective effects of antioxidants
against free radicles have attracted many scientists to investigate whether antioxidants
can be used to reverse ageing-associated functional decline and to improve the cellular
lifespan and longevity. The results have thus far been mixed. In radiation injury mod-
els, antioxidant agents have been shown to decrease oxidative stress and improve the
recovery of hematopoiesis following radiation injury, but these substances have not im-
proved the regenerative capacity of HSCs nor shown restorative effects on aged HSCs.
Radiation-induced HSC damage is mechanistically distinct from age-associated changes in
HSCs [100,102,142]
. Clinical trials with the supplementation of antioxidants did not prove
beneficial in reducing ageing-associated disease or improving life expectancy. Further
complicating the picture, it has been shown that the prolonged exposure to the exogenous
antioxidants could have deleterious effects on the endogenous antioxidant systems, and
some studies have shown that the long-term introduction of antioxidants in the diet actually
reduced the overall lifespan [143,144].
Studies in knock-out and transgenic mice intended to manipulate antioxidant enzymes,
including Cu/Zn superoxide dismutase (SOD), MnSOD, catalase, and glutathione peroxi-
dase, have largely failed to show changes in the lifespan of the animals despite the exposure
Antioxidants 2022,11, 1291 11 of 22
to extreme levels of oxidative stress [
141
,
145
]. Dyskeratosis congenita (DC) is a genetic
disorder disease characterized by the loss of telomerase activity and the accumulation of
DNA damage and is associated with reduced HSC regeneration ability and the decline in
several HSC biological functions, the hallmark of HSC ageing. Using the DC mouse model
(Dkc1
15
), researchers found that the ageing-associated changes in HSCs were associated
with high concentrations of ROS. However, the administration of NAC to Dkc1
15
mice for
one year was not able to reverse ageing-associated changes in HSCs [146].
In summary, HSCs have complex, highly conserved, and multi-faceted endogenous
antioxidant systems that protect and enhance HSC recovery in response to ROS and
oxidative stress injuries. Exogenous antioxidants and synergistic agents augment the
efficiency of the cellular endogenous systems, although this effect is likely transient and
has not been shown to definitively produce a longevity benefit. Genetically engineered
mouse models for the majority of antioxidant enzymes have not demonstrated changes in
the lifespan of the animals.
5. Role of Thioredoxin in HSC Ageing Biology
5.1. Overview of the Thioredoxin System
The thioredoxin system is one of the major cellular antioxidant proteins in mammals
and is highly evolutionarily conserved amongst aerobic organisms. The thioredoxin system
consists of thioredoxin (Trx1), thioredoxin reductase (TrxR), and nicotinamide adenine
dinucleotide phosphate (NADPH as an electron donor to recycle oxidized Trx to its reducing
form). Ultimately, the thioredoxin antioxidant system catalyzes electron flux from NADPH
through TrxR reductase to Trx, which then keeps cellular organic molecules (proteins, lipids,
and DNAs) in the reduced form (Figure 3). The thioredoxin system is not only responsible
for reducing proteins but also for restoring proteins to their native structure, thus providing
resistance against stress and maintaining cellular integrity [147].
Antioxidants 2022, 11, x FOR PEER REVIEW 12 of 23
Figure 3. Thioredoxin system. Trx in the oxidized form, Trx-S2, or the reduced form, Trx-(SH)2. In
the reduced state, Trx directly reduces disulfides in oxidized substrate proteins (Pro-S2). The oxi-
dation reaction is reversible and is maintained by thioredoxin reductase TrxR, which is sustained
by the electron donor NADPH. Created with BioRender.com, accessed on 19 June 2022.
5.2. Trx-1: One of the Few Antioxidants Shown to Extend Lifespan in Transgenic Mouse Models
The role of Trx-1 in maintaining cellular functions such as cell growth, cell survival,
and cell longevity has been extensively studied but remains incompletely defined. The
Kopf lab found that the thioredoxin system is critical for maintaining reducing power for
the synthesis of ribonucleotides critical to the process of T cell DNA biosynthesis [155].
The inactivating components of the thioredoxin system resulted in the impaired develop-
ment and activation of T cells and myeloid populations and increased cell replication
stress [155,156]. Furthermore, studies have investigated the association between Trx-1 and
lymphoid homing, migration, killing abilities, and cytotoxic granules release by NK cells
[157]. These studies demonstrated a significant role of Trx-1 in maintaining the health of
both hematopoietic and immune systems throughout the ageing process [155157].
With greater relevance to this review, the role of Trx-1 in biologic ageing and longev-
ity has been investigated by the Yodoi lab, who have successfully generated thioredoxin
(encoded by Trx1 gene) transgenic mice (Tg, act-TRX1+/0) by overexpressing human Trx1
cDNA driven by the β-actin promotor [158]. Interestingly, these Trx-1 Tg mice have shown
a 35% extension in the lifespan compared to the matched controls [159], and tg mice are
more resistant to various inflammatory and immune injuries [160162]. Furthermore, Trx-
1 Tg mice have shown higher telomerase activity, widely considered as a substitute for a
biological clock. These data suggest that Trx-1 has biologic functions beyond the well-
described redox homeostasis functions and may play an important role in processes such
as stem cell differentiation/renewal, cellular senescence, and the lifespan of the organism.
The Ikeno lab published the first long-term survival study to further examine the
effects of overexpressing Trx-1 on ageing and ageing-associated pathology. The Ikeno
team cloned the same human Trx-1 transgene described above to generate TRX1 transgene
Figure 3.
Thioredoxin system. Trx in the oxidized form, Trx-S2, or the reduced form, Trx-(SH)2. In the
reduced state, Trx directly reduces disulfides in oxidized substrate proteins (Pro
-S2). The oxidation
reaction is reversible and is maintained by thioredoxin reductase TrxR, which is sustained by the
electron donor NADPH. Created with BioRender.com, accessed on 19 June 2022.
Antioxidants 2022,11, 1291 12 of 22
Thioredoxin (Trx) has two main isoforms: Trx-1 and Trx-2. Trx-1 is the predominant
isoform and is primarily cytosolic but easily crosses cell and nuclear membranes [
137
]. Trx-1
translocates into the nucleus upon stress conditions or can be secreted extracellularly via a
unique leaderless mechanism. Trx-2 is restricted in mitochondria [
137
]. Our current review
focuses on Trx-1. Trx-1 is a 12-kD target-selective protein disulfide reductase with two Cys
residues in its conserved active site. Trx is a multifunctional protein and has a distinct
structure that facilitates this translocation to the nucleus in certain conditions as well as ex-
ports to the extracellular membrane to serve as proinflammatory
cytokines [148,149]
.
Trx-1
provides a reducing equivalent that sustains a variety of cell biological functions, including
immune cell survival and cell proliferation, and maintains cellular redox
homeostasis [150]
.
Compared to other known reducing systems in the cell, Trx-1 is the only protein that main-
tains the reducing power for the ribonucleotide reductase enzyme, which is the building
block for DNA replication and
repair [150,151]
. In addition to functioning as antioxidant,
the reduced form Trx-(SH)2 contains a dominant motif to catalyze proteins in a manner
similar to protein phosphorylation and dephosphorylation and can interact with a variety
of proteins, including transcription-binding factors at the genomic level [152154].
5.2. Trx-1: One of the Few Antioxidants Shown to Extend Lifespan in Transgenic Mouse Models
The role of Trx-1 in maintaining cellular functions such as cell growth, cell survival,
and cell longevity has been extensively studied but remains incompletely defined. The
Kopf lab found that the thioredoxin system is critical for maintaining reducing power for
the synthesis of ribonucleotides critical to the process of T cell DNA
biosynthesis [155]
.
The inactivating components of the thioredoxin system resulted in the impaired devel-
opment and activation of T cells and myeloid populations and increased cell replication
stress [155,156]
. Furthermore, studies have investigated the association between Trx-1
and lymphoid homing, migration, killing abilities, and cytotoxic granules release by NK
cells [157]
. These studies demonstrated a significant role of Trx-1 in maintaining the health
of both hematopoietic and immune systems throughout the ageing process [155157].
With greater relevance to this review, the role of Trx-1 in biologic ageing and longevity
has been investigated by the Yodoi lab, who have successfully generated thioredoxin
(encoded by Trx1 gene) transgenic mice (Tg, act-TRX1
+/0
) by overexpressing human Trx1
cDNA driven by the
β
-actin promotor [
158
]. Interestingly, these Trx-1 Tg mice have shown
a 35% extension in the lifespan compared to the matched controls [
159
], and tg mice are
more resistant to various inflammatory and immune injuries [
160
162
]. Furthermore, Trx-1
Tg mice have shown higher telomerase activity, widely considered as a substitute for a
“biological clock”. These data suggest that Trx-1 has biologic functions beyond the well-
described redox homeostasis functions and may play an important role in processes such
as stem cell differentiation/renewal, cellular senescence, and the lifespan of the organism.
The Ikeno lab published the first long-term survival study to further examine the effects
of overexpressing Trx-1 on ageing and ageing-associated pathology. The Ikeno team cloned
the same human Trx-1 transgene described above to generate TRX1 transgene mice (Tg,
act-TRX1
+/0
), but with a longer follow-up (29–30 months) under optimal conditions. They
found that the overexpression of TRX1 has benefits at both the cellular and organ levels,
and the lifespan extension occurs mainly in earlier ages with limited effects in later ages.
Additionally, young Trx-1 transgenic mice showed noticeable improvements in immune
function, inflammatory response, and the resistance to oxidative damage compared to the
control group. It was also noted that aged Trx-1 transgenic mice have shown a higher
incidence of total fatal tumors and fatal lymphoma compared to wild-type mice [163,164].
These findings demonstrated an important role of TXN1 in ageing, and Trx-1 is one of
the very few antioxidants that are found to extend the lifespan of transgenic mice.
Antioxidants 2022,11, 1291 13 of 22
5.3. Thioredoxin-1 Enhances HSC Functions in Animal Models of Hematopoietic Stem Cell
Transplant and Radiation Injury
The role of Trx-1 in the function and ageing of HSCs remains unclear. Using a
mass-spectrometry based semi-quantitative proteomics screen, our lab previously showed
that Trx-1 was significantly upregulated in the BM of hematopoietic stem cells trans-
planted (HSCT) recipient mice treated with AMD3100, also known chemokine receptor
type 4 (CXCR4) antagonist (plerixafor, trademark Mozobil), relative to the controls [
165
].
AMD3100 treatment has improved hematopoietic recovery following myeloablative HSCT
in our mouse model and in patients receiving myeloablative allogeneic transplants [
165
,
166
].
We have demonstrated the marked proliferative and protective effects on HSCs in animal
models of HSCT and radiation injury. We showed that the ex vivo culture of murine
HSCs with recombinant Trx-1 enhances their long-term repopulation capacity, reduces
cell senescence and radiation-induced double-strand DNA breaks, and downregulates
apoptotic-signaling pathways. We found that the administration of recombinant Trx-1 up
to 24 h following lethal total body irradiation (TBI) rescues BALB/c mice from radiation-
induced lethality [167].
It remains to be determined how AMD3100 increases Trx-1 expression. AMD3100 may
increase Trx-1 indirectly through other mediators in the niche microenvironment. For exam-
ple, studies have shown that AMD3100 attenuates the expression of Txnip, which directly
binds to the reduced form of Trx-1 and inhibits Trx-1 expression and activity [
168
,
169
].
Txnip
/
mice exhibited the overexpression of CXCR4/SDF-1, which was downregulated
by the administration of AMD3100 [168,169].
5.4. Emerging Evidence of Thioredoxin-1 in Protecting HSCs from Ageing
The protective effects of Trx-1 on HSCs in HSCT and in radiation injury led us to
determine the role of Trx-1 in HSC ageing. We first sorted by flow cytometry Lin
Sca-1
+
c-
Kit
+
CD150
+
CD48
long-term (LT)-HSCs, Lin
Sca-1
+
c-Kit
+
CD150
CD48
short-term (ST)
HSCs, Lin
Sca-1
+
c-Kit
+
CD150
CD48
+
multipotential progenitors (MPPs), and lineage+
cells from C57BL/6J mice aged at 3 weeks, 27 weeks, or 65 weeks; then, we measured
Trx-1 and p53 mRNA expression. Trx1 mRNA expression dramatically increases in the
LT-HSCs from 65-week-old mice compared to that from mice at a younger age. TP53 mRNA
expression noticeably reduced in the aged mice compared to the younger mice (Figure 4A,B).
To test whether Trx-1 plays a critical role in regulating HSC ageing, our lab crossbred
TXN1 conditional knockout mice (TXN
fl/fl
) [
170
] with ROSA-ER-Cre mice and generated
ROSA-ER-Cre-TXN
fl/fl
. ROSA-Cre-TXN
fl/fl
mice and control mice (either TXN
fl/fl
or
ROSA-ER-Cre mice) were treated with tamoxifen (75 mg/kg i.p. daily for 5 days), which
leads to the complete knockout of TXN1 in bone marrow samples at day 10 of injection.
Our unpublished data suggested that the deletion of TXN1 significantly attenuated the
reconstitutional, self-generating, and repopulating capacities of HSCs in lethally irradiated
recipient mice. Furthermore, HSCs lacking TXN1 have shown phenotypes characteristic
of aged HSCs, including the higher expansion and proliferation of LT-HSCs (unpublished
data). We measured the expression of genes that are associated with the ageing phenotypes
of HSCs, such as p16, p21, p38/MAPK, and Wnt5a [
36
,
171
]. The deletion of TXN1 in HSCs
upregulated the mRNA expression of p16, p21, p38/MAPK, and Wnt5a (Figure 4C). We
measured the peripheral leukocytes in the ROSA-Cre-TXN
fl/fl
mice and control mice at
different ages (2 months and 10 months old). The percentage of leukocytes in the peripheral
blood cells reduced by more than twofold after the deletion of TXN1 in young (2 months
old) mice compared to the age-matched control animals. No significant change in the
percentage of peripheral blood leukocytes was observed in old (10 months old) mice
(Figure 4D). Our data suggested that the role/contribution of Trx-1 in HSC ageing could be
different at different stages of the lifespan.
Antioxidants 2022,11, 1291 14 of 22
Antioxidants 2022, 11, x FOR PEER REVIEW 14 of 23
phenotypes of HSCs, such as p16, p21, p38/MAPK, and Wnt5a [36,171]. The deletion of
TXN1 in HSCs upregulated the mRNA expression of p16, p21, p38/MAPK, and Wnt5a
(Figure 4C). We measured the peripheral leukocytes in the ROSA-Cre-TXNfl/fl mice and
control mice at different ages (2 months and 10 months old). The percentage of leukocytes
in the peripheral blood cells reduced by more than twofold after the deletion of TXN1 in
young (2 months old) mice compared to the age-matched control animals. No significant
change in the percentage of peripheral blood leukocytes was observed in old (10 months
old) mice (Figure 4D). Our data suggested that the role/contribution of Trx-1 in HSC age-
ing could be different at different stages of the lifespan.
Figure 4. The loss of TXN1 enhances hematopoietic stem cells ageing: (A,B) Various hematopoietic
cell populations from wild-type mice C57Bl/6 at 3, 27, and 65 weeks of age were sorted and meas-
ured for TXN1 (A) and TP53 (B). (C) BM Lin cells from the ROSA-ER-Cre-TXNfl/fl mice and controls
were used to measure mRNA expression for Wnt5a, p21, p16, and p38. (D) The ROSA-ER-Cre-
TXNfl/fl mice and controls at 2 months or 10 months were treated with tamoxifen, and at 10 days,
peripheral leukocytes were measured. The data are represented as the mean ± SD. ** p < 0.01, *** p <
0.001.
5.5. Thioredoxin-1 Mediated Signaling Pathways in HSCs
Through ROS dependent and independent mechanisms, thioredoxin plays an im-
portant role in apoptotic regulation. In studies of murine thymoma cells (WEHI7.2), high
endogenous and exogenous Trx-1 was shown to be protective against both spontaneous
and drug-induced apoptosis [172]. Apoptosis regulator ASK1, a member of the MAPKK
family, activates apoptosis through a JNK-dependent pathway. A redox reaction involv-
ing Trx-1 has been shown to play a crucial role in this apoptotic pathway, with the over-
expression of Trx1 resulting in the increased reduction of ASK1, the impaired activation
of JNK, and, ultimately, decreased apoptosis with a minimal impact on the levels of ASK1
activation [173]. The reduced form of Trx-1 directly interacts with the N terminal of ASK1
and inhibits its kinase activity. Oxidative damage leads to oxidized Trx1, which
Figure 4.
The loss of TXN1 enhances hematopoietic stem cells ageing: (
A
,
B
) Various hematopoietic
cell populations from wild-type mice C57Bl/6 at 3, 27, and 65 weeks of age were sorted and measured
for TXN1 (
A
) and TP53 (
B
). (
C
) BM Lin cells from the ROSA-ER-Cre-TXN
fl/fl
mice and controls were
used to measure mRNA expression for Wnt5a, p21, p16, and p38. (
D
) The ROSA-ER-Cre-TXN
fl/fl
mice and controls at 2 months or 10 months were treated with tamoxifen, and at 10 days, peripheral
leukocytes were measured. The data are represented as the mean ±SD. ** p< 0.01, *** p< 0.001.
5.5. Thioredoxin-1 Mediated Signaling Pathways in HSCs
Through ROS dependent and independent mechanisms, thioredoxin plays an impor-
tant role in apoptotic regulation. In studies of murine thymoma cells (WEHI7.2), high
endogenous and exogenous Trx-1 was shown to be protective against both spontaneous
and drug-induced apoptosis [
172
]. Apoptosis regulator ASK1, a member of the MAPKK
family, activates apoptosis through a JNK-dependent pathway. A redox reaction involving
Trx-1 has been shown to play a crucial role in this apoptotic pathway, with the overex-
pression of Trx1 resulting in the increased reduction of ASK1, the impaired activation of
JNK, and, ultimately, decreased apoptosis with a minimal impact on the levels of ASK1
activation [173]. The reduced form of Trx-1 directly interacts with the N terminal of ASK1
and inhibits its kinase activity. Oxidative damage leads to oxidized Trx1, which dissociates
ASK1, and ASK1 then activates the SEK1-JNK and MKK3/MKK6-p38 signaling cascades to
induce program cell death. Additionally, Trx-1 promotes ASK1 ubiquitination and degra-
dation, and the association of Trx1/ASK1 could not be blocked by ROS or other apoptotic
signaling stimuli [
173
]. Trx-1 regulates NF-kappa B DNA binding affinity by the reduction
of a disulfide bond involving cysteine 62. Several lines of evidence have found that Trx1
regulates p53 activity.
5.6. Thioredoxin 2 and Mitochondrial Contributions to HSC Ageing
Recent studies have highlighted the significance of the mitochondrial regulation of
HSC quiescence [
174
]. Increase mitochondrial biogenesis is a key feature of the transition
of HSCs from quiescence to proliferation due to the increased energy requirement of active
Antioxidants 2022,11, 1291 15 of 22
HSCs. There is emerging evidence for an important mitochondrial checkpoint in the cell
cycle regulation of quiescence. Several mechanistic studies have begun to describe an
mTOR-LKB1-AMPK-dependent pathway regulation mitochondrial biogenesis, mitochon-
drial mass, and mitochondrial activity in the G
0
-to-G
1
cell cycle transition [
80
,
175
,
176
].
In the reverse order, it has been suggested that inadequate mitogenesis or a mitochon-
drial stress response (with contributing mitophagy) leads to a loss of HSC quiescence. If
mitochondrial stress is not adequately repaired, cell death occurs through apoptosis in
physiologic states. Although this review focuses primarily on Trx-1, Trx-2 is a mitochondrial-
restricted isoform of Trx with additional importance to HSC redox biology and ageing.
The specific inactivation of murine Trx2 is embryonically lethal in mice. Heterozygous
deletion results in a phenotype characterized by anemia, hepatic apoptosis, and signifi-
cantly reduced ex vivo hematopoiesis [
177
]. This finding suggests a critical role for the
thioredoxin system in HSC function and fitness through a mitochondrial restricted pathway
related to mitochondrial-mediated apoptosis, as described above. This mechanism has been
further characterized: in glutathione-depleted cells, Trx2 becomes oxidized, inactivated
Trx2 increases the susceptibility to TNF-alpha and ROS, and the overexpression of Trx2
protects against this toxicity [
178
180
]. Another unique aspect of mitochondrial Trx2 is that
Trx2 detoxifies ROS through mitochondrial specific peroxiredoxins (Prx3) and has its own
reductase (TrxR2) [179181].
In sum, the thioredoxin system consists of functionally distinct but synergistic cytosolic
Trx1 and mitochondrial Trx2 pathways, both regulating ASK1 activity and apoptosis.
6. Conclusions
As with all cells, HSCs regulate cellular functions through the apoptosis and senes-
cence pathways, with important implications for HSC ageing. As discussed above, a key
driver of HSC ageing is the accumulation of DNA damage through ROS exposure and
DDR errors. There is an evolutionary pressure to tightly balance the risk of malignant
transformation to hematologic malignancies with the importance of maintaining adequate
hematopoiesis throughout the lifespan. Complex cascades of molecular events and path-
ways including DNA damage response, ROS, mitochondrial regulatory mechanisms, and
apoptotic pathways work in concert to maintain this balance. Our data suggest that thiore-
doxin appears to play an important role in maintaining the biological functions of HSCs.
These findings provide a rationale and justification for further investigating the effects of
TXN1 as a molecule for mitigating against ageing-related changes in HSCs.
Funding:
This work is supported by the Duke Cancer Institute Fund (to Y.K.), NIH R01CA197792 (to
Y.K.), NIH R21CA234701 (to Y.K.), R21CA267275, and the American Society of Hematology Physician
Scientist Student Award (to P.M.).
Conflicts of Interest: All authors declare no competing conflict of interest.
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