Canonical Wnt Signaling in the Pathology of Iron Overload-Induced Oxidative Stress and Age-Related Diseases

Abstract

Iron accumulates in the vital organs with aging. This is associated with oxidative stress, inflammation, and mitochondrial dysfunction leading to age-related disorders. Abnormal iron levels are linked to neurodegenerative diseases, liver injury, cancer, and ocular diseases. Canonical Wnt signaling is an evolutionarily conserved signaling pathway that regulates many cellular functions including cell proliferation, apoptosis, cell migration, and stem cell renewal. Recent evidences indicate that iron regulates Wnt signaling, and iron chelators like deferoxamine and deferasirox can inhibit Wnt signaling and cell growth. Canonical Wnt signaling is implicated in the pathogenesis of many diseases, and there are significant efforts ongoing to develop innovative therapies targeting the aberrant Wnt signaling. This review examines how intracellular iron accumulation regulates Wnt signaling in various tissues and their potential contribution in the progression of age-related diseases.

Full text

Review Article
Canonical Wnt Signaling in the Pathology of Iron Overload-
Induced Oxidative Stress and Age-Related Diseases
Austin Armstrong ,
1,2
Ashok Mandala,
1,2
Milan Malhotra,
1,2
and Jaya P. Gnana-Prakasam
1,2
1
Department of Ophthalmology, Saint Louis University, St. Louis, USA
2
Department of Biochemistry & Molecular Biology, Saint Louis University, St. Louis, USA
Correspondence should be addressed to Jaya P. Gnana-Prakasam; jaya.gnanaprakasam@health.slu.edu
Received 15 December 2021; Accepted 4 January 2022; Published 25 January 2022
Academic Editor: Alin Ciobica
Copyright © 2022 Austin Armstrong et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
Iron accumulates in the vital organs with aging. This is associated with oxidative stress, inflammation, and mitochondrial
dysfunction leading to age-related disorders. Abnormal iron levels are linked to neurodegenerative diseases, liver injury, cancer,
and ocular diseases. Canonical Wnt signaling is an evolutionarily conserved signaling pathway that regulates many cellular
functions including cell proliferation, apoptosis, cell migration, and stem cell renewal. Recent evidences indicate that iron
regulates Wnt signaling, and iron chelators like deferoxamine and deferasirox can inhibit Wnt signaling and cell growth.
Canonical Wnt signaling is implicated in the pathogenesis of many diseases, and there are significant efforts ongoing to
develop innovative therapies targeting the aberrant Wnt signaling. This review examines how intracellular iron accumulation
regulates Wnt signaling in various tissues and their potential contribution in the progression of age-related diseases.
1. Introduction
A multitude of age-related diseases are associated with dis-
rupted cellular iron homeostasis. Wnt/β-catenin signaling,
also known as canonical Wnt signaling, is a crucial pathway
that mediates cell development and proliferation. Hence,
abnormal Wnt signaling can be detrimental and is implicated
in diseases across different tissue types. Interestingly, an
overlap between dysregulated iron homeostasis and aberrant
Wnt signaling exists in pathologies including colorectal
carcinoma (CRC) [1–3], diabetic retinopathy (DR) [4, 5],
and age-related macular degeneration (AMD) [6–8]. For this
reason, there has been a recent interest in studying the inter-
play between iron homeostasis and canonical Wnt signaling.
Iron is a necessary dietary component, as it is crucial to
processes ranging from oxygen transport by red blood cells
[9] to fetal neurodevelopment [10]. Cellular iron homeosta-
sis and mitochondrial iron homeostasis are interdependent
as mitochondria must import iron to form iron–sulfur clus-
ters and heme, which are critical for vital cellular functions.
Imbalances in iron homeostasis leading to its accumulation
can be particularly dangerous, such as in hereditary hemo-
chromatosis [11]. The toxicity due to iron overload is largely
related to the potential of iron to induce oxidative stress,
mitochondrial dysfunction, and inflammation. Iron-
mediated oxidative stress arises because excess iron, a proox-
idant, generates reactive oxygen species (ROS) via the
Fenton reaction [12]. ROS can then affect the integrity of
DNA [13], as well as other important proteins and lipids
involved in cellular function [14]. Iron-mediated mitochon-
drial dysfunction is a result of iron-induced mitochondrial
DNA damage, which correlates with defects in iron–sulfur
cluster biogenesis, electron transport chain, and heme
synthesis [15]. Iron-mediated inflammation occurs due to
the proinflammatory nature of both excess labile iron [16]
and iron bound to ferritin [17, 18], an iron storage protein.
Cellular labile iron promotes inflammation via interleukin-
1β(IL-1β) secretion and NLRP3 inflammasome stimulation
in human monocytes through the NF-κB pathway [16]. Sim-
ilarly, ferritin has been shown to function as a local cytokine
Hindawi
Oxidative Medicine and Cellular Longevity
Volume 2022, Article ID 7163326, 13 pages
https://doi.org/10.1155/2022/7163326

through NF-κB pathway activation in activated rat stellate
cells, leading to significant increases in inflammatory mole-
cules such as IL-1β[17]. Hyperferritinemia, a condition
characterized by excess ferritin levels, is constitutively linked
to disorders like familial hemophagocytic lymphohistiocyto-
sis [17] and macrophage activation syndrome [18] that are
characterized by overactive inflammatory responses. In
addition, research has demonstrated that inflammation itself
can lead to iron accumulation in tissues such as the retina
[19] and the liver [20] by upregulating hepcidin and thereby
decreasing ferroportin expression. These findings indicate a
positive feedback loop, where iron-mediated inflammation
leads to further iron accumulation and subsequently
increased iron-mediated inflammation, oxidative stress,
and mitochondrial dysfunction.
The rationale for much of the research discussed in the
following sections is that oxidative stress [21–23], inflamma-
tion [24, 25], and mitochondrial dysfunction [26] have also
been indicated as mediators of abnormal canonical Wnt
signaling. This overlap raises the fundamental question of
our review: what role does iron overload have in regulating
Wnt signaling? While the relationship between iron, Wnt
signaling, and cancer has become increasingly well-
established [27–29], this same relationship is less docu-
mented in pathologies outside of cancer. The present review
is aimed at providing an update on the canonical Wnt path-
way with respect to iron-mediated aberrant Wnt/β-catenin
signaling in several tissue-specific contexts.
2. The Canonical Wnt Pathway
2.1. Contextualizing Wnt. The first Wnt gene was discovered
in 1982 as an oncogene activated in mouse models of virally
induced mammary carcinoma and was named Int-1 [30].
Several years later, an important gene in Drosophila larval
development known as the wingless (Wg) gene was found
to be a homolog of the murine Int-1 [31]. The discovery of
the Wg homolog to mouse Int-1 and subsequent research
[32, 33] has demonstrated that the canonical Wnt pathway
is highly conserved amongst a variety of species. Even com-
plexity within the Wnt gene family seems to be conserved, as
the sea anemone Nematostella vectensis shares 11 of the 12
known Wnt gene subfamilies with humans [32]. Because of
the conserved nature of the Wnt genes and the implication
of Wnt regulation in a variety of physiological events,
Wnt/β-catenin research has led to a plethora of discoveries
over the last four decades.
Today, we know of 19 Wnt genes in the mammalian
genome that encode 12 subfamilies of Wnt proteins (Wnts)
[34]. These Wnts are involved in a range of cell events but
centrally act as growth factors and cause cell proliferation
in a variety of cell types during both embryogenesis and
adult tissue homeostasis [35–37]. In addition, Wnts contrib-
ute to the directional organization of proliferating cells by
altering gene expression and affecting cytoskeletal and
mitotic architecture [38–43]. All Wnt genes encode glyco-
proteins that are about 40 kDA in size and that contain
many conserved cysteine residues [44]. After translation,
important palmitoylation and glycosylation events occur
on Wnt proteins in the endoplasmic reticulum (ER). Palmi-
toylation involves the attachment of a palmitoleic acid to a
conserved serine residue on Wnt proteins [45, 46] by an
O-acyltransferase known as porcupine [47, 48]. This lipid
modification is necessary for Wnt signaling, as it is used
downstream as a binding site for the Wnt receptor, Frizzled
[49], as well as the Wntless protein that aids in Wnt secre-
tion [50, 51]. Of the two major types of posttranslational
modifications in the ER, glycosylation is the one less under-
stood. Wnts vary in the number of glycosylated sites present,
and the presence of these glycans seems to have varying
importance. Site-directed mutagenesis of specific glycosyl-
ated regions impairs secretion in certain Wnts [52], while
in other Wnt isoforms, it does not seem to have any affect
at all [53]. Though further studies are required to determine
the exact function of posttranslational Wnt glycosylation,
glycosylation currently has significance as a signal for the
secretion of few specific Wnt glycoproteins.
2.2. Wnt Trafficking and Secretion. The trafficking and secre-
tory pathways are two examples of conserved characteristics
between the human Wnt proteins [54, 55]. As aforemen-
tioned, Wnt trafficking and secretion are dependent upon
proper lipid modification by porcupine in the ER [47, 48].
This is because Wntless (Wls), a transmembrane protein,
binds the palmitoleic acid in the ER for proper Wnt traffick-
ing and subsequent Wnt release [50, 51, 56]. Indeed, knock-
outs of Wls or inhibitors of porcupine result in the
accumulation of Wnt proteins in ER [57]. Once bound to
Wnt, Wls will travel with Wnt through the Golgi and ulti-
mately to the plasma membrane [50, 51, 56]. Here, Wls
may act in one of the two ways. One possibility is that
Wls can leave its respective Wnt glycoprotein and return
to the trans-Golgi network [58–60]. This occurs via retro-
grade movement involving clathrin-mediated endocytosis
and retromer retrieval mediated by a specialized ER-
retrieval motif [50, 57, 61, 62]. Alternatively, Wls may be
incorporated into the membrane of an exosome along with
Wnt for secretion [63, 64].
The distance of Wnt secretion and subsequent signaling
is a heavily debated topic, as there is evidence for Wnt target-
ing both local [34, 65] and distant [66, 67] cells. At the Wnt
secreting membrane, varying events can occur depending
on the desired distance of secretion. Wnts can be tethered
for contact-dependent interactions with nearby cells [68]
or used for longer range signaling either through expres-
sion on an exosome with Wntless requiring R-SNARE
Ykt6 activity [69] or by binding to solubilizing molecules,
such as Swim [66]. After Wnt trafficking and secretion,
signaling occurs. The known Wnt pathways can be orga-
nized into single canonical Wnt pathway and two
noncanonical Wnt pathways [37]. This review will focus
on the canonical Wnt pathway, which involves the nuclear
localization of active β-catenin, as this is the best studied
pathway in relation to iron homeostasis.
2.3. Wnt/β-Catenin Signaling Cascade. In canonical Wnt
signaling, there is a complex system of transducing factors
that cooperate to instill cell responsiveness to the Wnt
2 Oxidative Medicine and Cellular Longevity

ligands. A central player in this cascade is β-catenin, as it is
the primary regulator in Wnt target gene expression. With
cytosolic accumulation of β-catenin, subsequent nuclear
translocation occurs allowing the protein to bind to the T-
Cell Factor/Lymphoid Enhancer Factor (TCF/LEF) tran-
scription factor family [70, 71]. Wnt target cells exhibit the
surface receptors Frizzled (FZD) and low-density
lipoprotein-related receptor protein 5 or 6 (LRP5/6) to initi-
ate the signal cascade [34]. FZD is a family of cell surface
proteins with seven transmembrane domains that contain
both an extracellular N-terminal cysteine-rich domain that
interacts with Wnt [49, 72, 73], as well as a hydrophobic
groove that binds the lipid modifications on Wnt [49].
LRP5 and LRP6 serve as coreceptors necessary for Wnt
transduction that form heterodimers with FZD in the pres-
ence of Wnt [74, 75]. It has been posited that LRPs contain
binding regions for Wnt, as described by several anti-LRP
monoclonal antibody studies [76]. While there are a variety
of other Wnt receptors that are important for noncanonical
signaling [77], the FZD and LRP-mediated pathway will
remain the focus of this review as it is the best understood
with relation to iron.
In the absence of Wnt, cytoplasmic β-catenin is bound
by an intact destruction complex and targeted for
ubiquitin-mediated degradation, preventing β-catenin accu-
mulation and nuclear translocation. This ultimately results
in Wnt target gene repression. The destruction complex
consists of proteins Axin, Adenomatous polyposis coli
(APC), and Wilms tumor gene on the X chromosome
(WTX), casein kinase 1-α(CK1α), glycogen synthase kinase
3-β(GSK-3β), and other factors [34]. Axin is a scaffolding
protein that interacts with β-catenin and other members of
the destruction complex [34]. APC and WTX, two tumor
suppressor proteins, are also essential for an effective
destruction complex [34]. CK1αand GSK-3βare serine-
threonine kinases that perform multiple functions related
to Wnt signaling. In terms of the destruction complex, they
phosphorylate Axin-bound β-catenin to target it for ubiqui-
tination [34, 78]. β-Transducin repeat-containing protein
(β-TrCP), a component of the E3 ubiquitin ligase complex,
recognizes phosphorylated β-catenin and catalyzes the addi-
tion of ubiquitin polymers to direct β-catenin for degrada-
tion by proteasomes preventing their nuclear translocation
[78]. The absence of β-catenin in the nucleus leaves the
repressive TCF/LEF complex active, which then permits
Groucho/transducin-like enhancer (Gro/TLE) family
proteins to recruit histone deacetylases that inhibit Wnt
target gene expression [79].
In the presence of Wnt, canonical Wnt stimulation
begins with Wnt ligand facilitating the heterodimerization
of FZD and its coreceptor, such as LRP5 or LRP6. Ligation
induces a conformational change in the receptors and by a
currently unclear mechanism, recruits, and activates GSK-
3βand CK1γ[80–82]. At the membrane, GSK-3βand
CK1γphosphorylate the LRP tail at the PPPSP motif [83]
and in regions adjacent to the PPPSP motifs [84], respec-
tively. This sequential phosphorylation of the LRP tail
results in Axin relocation to the plasma membrane and its
physical removal from the cytosol, inhibiting the formation
of the β-catenin destruction complex [34]. β-Catenin then
accumulates in the cytosol and subsequently translocates to
the nucleus. Nuclear β-catenin interacts with the TCF/LEF
interface discussed earlier to activate Wnt target gene
expression [70, 71, 85, 86] as shown in Figure 1.
2.4. Wnt/β-Catenin Modulation. As previously discussed,
Wnt/β-catenin signaling plays a multitude of roles in a host
of tissues throughout life. Wnts generally function as growth
factors to cause proliferation and have more than 100 down-
stream target genes [35–37, 87]. Many of these target genes
are important cell cycle regulators including C-Myc [88]
and Cyclin D [89]. Others include those related to angiogen-
esis like VEGF [90], as well as nearly every component of the
Renin-Angiotensin-System (RAS) [91]. Wnt signaling acti-
vates mitochondrial biogenesis, in turn producing elevated
levels of ROS and oxidative damage [26], which is consid-
ered to be the cause for certain pathological consequences.
Given the wide-ranging functions of canonical Wnt signal-
ing, it is logical that dysfunctional Wnt/β-catenin signaling
would have a variety of negative implications. The nature
of this aberrance, however, is not as intuitive. In some
diseases, such as in cases of CRC [92] or AMD [7, 8], Wnt
signaling is pathologically upregulated, while in others, such
as in Norrie disease [93] or osteopenia [94], Wnt signaling is
pathologically downregulated. For this reason, a variety of
positive and negative modulators exist and are important
for the understanding of Wnt signaling.
2.4.1. Positive Modulation. Positive modulators of Wnt/β-
catenin signaling are those that lead to an increase in active
β-catenin translocating to the nucleus and thus increasing
the expression of Wnt target genes. In cases of chronic
wounds [95], vitiligo [96, 97], or other diseases character-
ized by Wnt/β-catenin inactivation, positive Wnt modula-
tors can be protective. However, in the context of diseases
characterized by Wnt/β-catenin activation, positive Wnt
modulators may be harmful. For example, patients being
treated with lithium, a canonical Wnt activator, were
shown to have a significantly increased chance of develop-
ing renal carcinogenesis [98].
One important positive regulation pathway of vertebrate
Wnt signaling involves the four R-spondin (RSPO) proteins,
which are characterized by two furin domains and a throm-
bospondin domain [99]. In the absence of RSPO ligands, the
disheveled protein promotes the destruction of FZD on the
membrane by the E3 ligases [100]. However, in the presence
of RPSO proteins, the RSPO ligands will bind to leucine-rich
repeat-containing G protein-coupled 5 (Lgr5) family recep-
tors [101–103] resulting in the membrane clearance of E3
ligases RNF43 and ZNRF3 [104, 105]. In effect, this pro-
motes FZD accumulation on the membrane and increases
the sensitivity of a cell to Wnt ligands [104, 105]. Norrin is
a second example of a secreted protein that positively
modulates Wnt signaling [106]. It is characterized by its
cysteine-knot motif [107] and, with the help of its coreceptor
tetraspanin 12 [108, 109], acts by binding directly to the
FZD4/LRP5 complex to activate the canonical Wnt pathway
[93, 110, 111].
3Oxidative Medicine and Cellular Longevity

In addition to the well-studied RSPO and Norrin posi-
tive modulators, there are several others that have been dis-
covered over the years. Protein phosphatase-2A (PP2A) is a
serine-threonine phosphatase composed of three subunits
that can positively regulate the canonical Wnt pathway by
dephosphorylating a variety of proteins including β-catenin
[112]. Other examples include microRNAs miR-135a and
miR-135b that directly repress APC expression in colorectal
cancer cells, thereby destabilizing the β-catenin destruction
complex and allowing Wnt target gene expression [113].
Still, others like heparin sulfate proteoglycans serve as cofac-
tors to promote Wnt signaling in the control of C. elegans
mitotic spindle orientation [114], distal-tip cell migration
[115], and neuronal positioning [116].
2.4.2. Negative Modulation. Negative modulators of Wnt/β-
catenin signaling are those that lead to the destruction of β-
catenin and a decrease in the expression of Wnt target genes.
This may be protective in diseases characterized by Wnt/β-
catenin activation, but deleterious during diseases character-
ized by Wnt/β-catenin inactivation [117]. The potentially
deleterious effect of negative modulators can be seen in the
progression of Alzheimer’s disease, a disease characterized
by Wnt inactivation [118]. Specifically, the worsening of
amyloid beta plaque accumulation and subsequent synaptic
loss was observed with the presence of the negative regulator
Dickkopf-1 [119, 120].
A prototypical downregulator of Wnt signaling is
Notum, an extracellular enzyme that inhibits Wnt proteins
by removing their vital palmitoylate residues [121, 122].
Notum also partakes in a negative feedback loop, as the
TCF/LEF family transcription factors have binding sites in
the Notum promoter region to increase its expression [121,
123]. In addition to Notum, extracellular Wnt inhibitors
include Wnt inhibitory factor (WIF) proteins [124, 125]
and secreted FZD-related proteins (SFRPs) [126–128] that
directly bind Wnt proteins to inhibit signaling. There is also
another important class of negative Wnt modulators that act
as transmembrane antagonists by binding and subsequently
blocking Wnt receptors from interacting with Wnt. These
include Shisa [129], Wnt-activated inhibitory factor [130],
Adenomatosis polyposis coli downregulated 1 [131], and
Tiki1 [132]. Lastly, miRNAs have been a recent area of
research that have been implicated for negatively modulat-
ing Wnt signaling. Some examples include miR-200a, which
decreases Wnt target gene transcription [133], miR-21,
which directly represses Wnt1 protein production [134],
and miR-184, which targets Wnt coreceptor FZD-7 to pre-
vent Wnt signaling in retinal neovascularization [135].
In addition to the Notum feedback loop, there are several
other negative feedback loops that regulate canonical Wnt
signaling. An important one of these was mentioned in our
discussion of positive Wnt regulation, with regard to the
E3 ligases RNF43 and ZNRF3. These two ligases are known
to control FZD membrane expression by mediating the
ubiquitination of the FZD cytoplasmic loops, which leads
to FZD lysosomal degradation [136, 137]. A third canonical
Wnt signaling negative feedback loop is the conductin/
Axin2 loop. Axin2 is a Wnt target gene, and its expression
mimics Axin in the destruction complex to increase β-
catenin degradation [138, 139], which subsequently downre-
gulates Wnt target gene expression.
2.5. Iron-Mediated Wnt/β-Catenin Signaling during
Pathological Conditions. In the remainder of this review,
we will discuss the recent findings on how iron modulates
APC Axin
CK1αGSK3β
L
R
P
6
Frizzled
Active destruction complex
APC
Axin GSK3β
CK1α
β-catenin
Frizzled
Wnt
ligands
Fe
Fe
Fe
Fe
Fe
??
p
Inactive destruction complex
β-catenin
pp
Degraded β-catenin
oxidative
stress
β-catenin
TCF/LEF
Axi n2
cMYC
CCND
Renin angiotensin system
OFF STATE ON STATE
Liver Retina
L
R
P
6
Figure 1: A schematic overview of tissue-dependent Wnt signaling during conditions of iron overload.
4 Oxidative Medicine and Cellular Longevity

Wnt/β-catenin signaling leading to multiple disorders as
outlined in Figure 2.
2.5.1. Cancer. The role of Wnt signaling in cancerous pathol-
ogies is historically validated, with the discovery of Wnt
signaling based on experimentation involving mouse models
of breast cancer and cancer-causing retrovirus mouse mam-
mary tumor Virus (MMTV) [140]. In experiments that
lasted until the 1990s, MMTV was found to insert proviral
DNA into specific regions of the mouse genome, inducing
oncogene formation and mammary hyperplasia, and these
genes were all later connected to Wnt signaling [141–143].
In addition, the growing clinical significance of Wnt signal-
ing is also closely related to cancer, as the most well-known
pathology that involves Wnt is familial adenomatous poly-
posis (FAP) [144]. In FAP, mutation in APC and stabiliza-
tion of β-catenin results in increased colonic cell
proliferation, yielding a presentation of colonic polyps asso-
ciated with increased colorectal cancer risk [145–147]. In
addition to MMTV studies and FAP being related to cancer,
recent research has demonstrated that Wnt contributes to
gastrointestinal, hematopoietic, breast, skin, brain, and
colonic cancers [144].
With researchers investigating more on the connection
between cancer and Wnt, iron has emerged as a prominent
Wnt regulator in the context of proliferative pathologies.
In studies using APC knockout cell lines Caco-2 and
SW480, it was highlighted that growth on FeSO4-loaded
media and hemin-loaded media, both rich in iron, increased
Wnt signaling [27]. Thus, iron-mediated Wnt signaling
upregulation was demonstrated in cell lines that resembled
FAP-associated cancer cells with the presence of APC
knockout [27]. Another recent study that investigated Nrf2
mutations and their association with hepatocellular carci-
noma reported that alterations in iron homeostasis and
subsequent Wnt signaling activation play a role in the occur-
rence and proliferation of hepatocellular carcinoma [148].
These studies show a potential contribution of iron in Wnt
upregulation during cancerous pathologies.
Two recent reports revealed that iron chelators can
reverse the Wnt activation during cancer. In the first study,
a specific iron chelator HQBA inhibited Wnt signaling in a
variety of cancer cell lines and inhibited growth of
mammary tumors in MMTV-Wnt1 mouse models of Wnt-
dependent breast cancer and in MMTV-PyMT mouse
models of Wnt-independent breast cancer [29] indicating
the plausible proliferative effects of iron both dependent
and independent of Wnt signaling. The significance of this
study with relation to iron is garnered from the fact that
HQBA premixed with iron prior to cancer cell line exposure
prevented its ability to reverse Wnt/β-catenin activation in
several tissue-specific cancer cell lines confirming that the
antitumor effect of HQBA is through iron chelation [29].
A second study identified acyl hydrazones as inhibitors
of Wnt/β-catenin signaling by chelating iron. Upon treat-
ment with acyl hydrazones, intracellular iron was chelated,
Wnt/β-catenin activation was reversed, and cell prolifera-
tion significantly decreased in human CRC cell lines
SW480 and DID-1 [149]. Taken together, these studies
strongly suggest that excess iron upregulates Wnt/β-
catenin signaling in certain cancerous pathologies, and
that iron chelation may be a potential therapeutic strategy
to prevent cancer progression.
2.5.2. Neurodegenerative Disorders. The relationship
between iron overload and aberrant Wnt signaling has been
described in two studies that suggest excess iron in neuronal
cells pathologically increases Wnt/β-catenin signaling. The
first study discusses posthemorrhagic chronic hydrocephalus
(PHCH), a potentially fatal medical condition often arising
after an intraventricular hemorrhage (IVH) [150]. PHCH
is known to cause an increase in both cerebral spinal fluid
(CSF) iron concentration [151] and ferritin content within
the brain [152]. Additionally, PHCH is characterized by
fibrotic changes, which, in other tissues, are known to be
linked to dysregulated Wnt/β-catenin signaling [153, 154].
As iron accumulation and fibrotic changes are both major
players in the progression of PHCH, authors investigated
the therapeutic ability of the iron chelator deferoxamine
(DFX) in the treatment of abnormal Wnt signaling in a
PHCH model. The study revealed that by chelating excess
iron from CSF and brain ferritin, DFX normalized the
upregulated Wnt/β-catenin signaling seen in PHCH after
an IVH. This served to broadly improve PHCH occurrence
and severity [155]. Similarly, another recent study reported
that upregulation of Wnt/β-catenin signaling in neural pro-
genitor cells (NPCs) could be normalized by DFX, leading to
an increase in NPC differentiation and outgrowth [156].
These studies indicate that iron overload is detrimental to
the cells in neural tissues due to Wnt/β-catenin activation,
which can be therapeutically resolved with DFX treatment.
2.5.3. Bone Remodeling. Iron [157, 158] and canonical Wnt
signaling [159] are both important players in the mainte-
nance of bone. While dietary iron is critical for healthy bone
density in populations such as postmenopausal women
[157], iron overload inducing diseases like β-thalassemia
and hemochromatosis are often associated with decreased
bone density and integrity [160]. Indeed, chelation of iron
with deferasirox has been shown to improve bone density
in patients with β-thalassemia [161], suggesting that excess
iron is detrimental to bone health. On the other hand,
normal Wnt signaling is crucial for appropriate bone remod-
eling because Wnt signaling is involved in both osteoblast
differentiation and osteoclastogenesis [159, 162, 163]. In
addition, osteoporosis, a disease characterized by reduced
bone density due to ineffective bone remodeling [164], is
associated with both decreased Wnt signaling and oxidative
stress [165]. For these reasons, a recent article investigated
iron-dependent Wnt signaling in bone marrow stromal cells
differentiated towards osteoblasts [166]. The study found
that excess iron is detrimental to osteoblast differentiation,
and that iron chelation using DFX can reverse the negative
effects of iron overload in the same cells. Moreover, the
results conclude that induction of Wnt5a expression by
DFX is the mediator of this recovery, which occurs through
the PI3K and NFAT pathways [166]. These results indirectly
indicate that excess iron reduces Wnt expression in bone;
5Oxidative Medicine and Cellular Longevity

iron chelation by DFX treatment induces Wnt5a expression
to recover the Wnt signaling. However, the study neither
demonstrated directly that iron overload decreases Wnt sig-
naling, nor showed that chelation of iron brings back Wnt
signaling in the osteoblasts. A final consideration is that
Wnt5a operates through a noncanonical Wnt pathway
[167]. Despite these limitations, we can still conclude that
the induction of Wnt through DFX treatment is therapeutic
for iron-overloaded osteoblasts. Moreover, recent research
on an in vivo iron-induced osteoporotic rat model demon-
strated similar reduction of Wnt signaling that was recov-
ered with DFX in bone tissues [168]. Another in vivo
mouse study suggests that hepcidin-induced osteoporosis,
mediated through iron overload, may also be targeted via
inhibition of Forkhead box O3a to recover canonical Wnt
signaling [169].
Interestingly, while iron overload leads to decreased
activity of the Wnt pathway in osteoblasts, another study
reported that iron overload induces ROS-mediated apopto-
sis and upregulation of the Wnt pathway in bone marrow
mesenchymal cells of patients with myelodysplastic syn-
dromes [170]. This highlights the variation in Wnt signaling
in response to iron overload between different cell types even
within the context of a single tissue like bone.
2.5.4. Liver Injury. Chronic iron overload in hepatocytes,
such as in cases of hemochromatosis, is associated with
severe hepatic injury and cancer largely because of iron-
induced steatosis, fibrosis, inflammation, and oxidative
stress [171, 172]. Interestingly, liver-specificβ-catenin
knockout (KO) mice are known to have similar hepatic
pathology resulting from factors such as fibrosis and oxida-
tive stress [173, 174]. For these reasons, a recent study inves-
tigated the role of iron overload in hepatic pathology of
liver-specificβ-catenin KO mice [175]. This study con-
cluded that following iron overload, liver-specificβ-catenin
KO mice have increased steatohepatitis and fibrosis that is
preceded by both inflammation and oxidative stress. More-
over, they demonstrate that treatment with an antioxidant
can help prevent this disease progression. In effect, the
absence of β-catenin exacerbates the detrimental effects of
hepatic iron overload. The study also showed that iron over-
load led to a decrease in β-catenin in mice where it was not
knocked out. This is postulated to be an initial protective
mechanism, because these mice had less expression of
Cype2e1, a β-catenin target associated with oxidative stress
[175]. However, the delicate nature of Wnt/β-catenin
expression was proven to be detrimental in cases of β-
catenin KO which increased inflammation and eventually
lead to Cype2e1 reappearance, and, thus, further decrease
in β-catenin due to prolonged iron overload could prove to
be similarly detrimental in liver tissue. We recently showed
that iron overload in normal mouse liver decreases Wnt
pathway by suppressing Sirtuin 3 signaling [176]. Thus,
studies so far indicate that iron overload downregulates
Wnt signaling in liver contributing towards the progression
of liver fibrosis.
2.5.5. Ocular Diseases. Visual impairment is a national and a
global health concern that impairs physical and mental
health in affected individuals. The retina is one of the highest
energy consuming organs in the body. Impaired metabolic
changes in eye diseases often drive neuronal and vascular
pathologies [177, 178]. The most prevalent retinal degenera-
tive diseases are age-related macular degeneration and
diabetic retinopathy. Identification of risk factors and the
molecular mechanisms that govern neuronal cell loss and
vascular changes is of interest for translational researchers
and clinicians to discover preventive and interventional
therapeutics for retinal disorders of the eye.
Retina expresses many iron containing proteins like
RPE65, an isomerohydrolase that converts all-trans-retinyl
ester to 11-cis-retinol in the visual cycle [179], fatty acid
desaturase, an enzyme involved in synthesis of membrane
lipids 9 [180], and guanylate cyclase, involved in the synthe-
sis of cGMP, a second messenger in the phototransduction
pathway. So iron is critical for the retinal health. However,
excess iron due to its prooxidant property induces oxidative
stress [181] resulting in impaired retinal function. We have
previously shown that excess iron alters retinal barrier integ-
rity and accelerates the retinal cell loss by augmenting oxida-
tive stress and inflammasome activation [4]. Retinal iron
accumulation has been reported in the human patients and
mouse models of AMD and DR [4, 6]. Similarly, multiple
Neurodegenerative
diseases
Tumor cell
proliferation
AGING
Bone remodeling
Liver injury
Ocular disorders
IRON OVERLOAD
Fe
Fe
Fe
FeFe
Fe
Fe
Fe
Fe
Fe
Fe
Fe
Fe
Aberrant Wnt/β-catenin signaling
Fe
Fe
Fe
Fe
Fe
Fe
Fe
Figure 2: Iron accumulation associated with aging modulates canonical Wnt/β-catenin signaling leading to the progression of liver injury,
neurodegenerative diseases, bone remodeling, cancer, and ocular disorders.
6 Oxidative Medicine and Cellular Longevity

studies have confirmed the pathogenic role of canonical Wnt
signaling in the etiology of AMD [7, 8, 182] and DR [5, 183].
Recently, we reported a critical role for iron-induced oxida-
tive stress in the activation of canonical Wnt pathway medi-
ated by peroxisome proliferator-activated receptor- (PPAR-)
alpha signaling in retina [184]. A comprehensive under-
standing of what drives iron overload and the downstream
Wnt signaling in an increasingly wide range of diseases
would help in preventing the progression of diseases at an
earlier stage in the future.
3. Conclusion
A multivariate genomic scan has revealed high levels of iron
in the blood to be intimately associated with reduced health-
span [185]. Also, intake of iron-rich diet and excess iron
supplements are implicated in many age-related disorders.
Iron-mediated oxidative stress induces inflammation and
mitochondrial dysfunction thereby playing a critical role in
the progression of cancer, neurodegenerative disorders, oste-
oporosis, liver fibrosis and steatosis, and ocular diseases.
Similarly, aberrant changes in the canonical Wnt/β-catenin
signaling pathway are a hallmark of cancer, diabetes melli-
tus, and other degenerative disorders. Elucidating the com-
plex interplay between iron and Wnt pathway could lead
to new insights into the mechanisms of disease progression
and enrich our understanding of the aging biology. Control
of body iron stores and Wnt inhibitors can thus serve as
promising clinical targets to overcome the myriad ramifica-
tions of aging.
Data Availability
Data supporting this Review are from previously reported
studies, which have been cited.
Conflicts of Interest
The authors declare that they have no competing interests.
Authors’Contributions
A.A., A.M., M.M., and J.P.G wrote, edited, and finalized the
manuscript. Austin Armstrong and Ashok Mandala contrib-
uted equally to this work.
Acknowledgments
The financial support from the National Eye Institute (R01-
EY031008) and the American Heart Association
(14SDG20510062) is kindly acknowledged.
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