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HIF-1α’s role in chronic liver diseases and neurodegenerative diseases
Cheryl Cheah*
Department of Biochemistry and Chemistry, University of Arizona, Tucson, Arizona
85721, United States
Abstract Word Count: 154 words
Main Body Word Count: 2966 words
*Corresponding author
Cheryl Cheah, Department of Biochemistry and Chemistry, University of Arizona,
Tucson, Arizona 85721, United States.
Phone: 480-612-3046, email: [email protected]
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Abstract
HIF-1α is a transcription factor that activates downstream effects in metabolic pathways
and vascular pathways. In normoxic conditions, HIF-1α is targeted for ubiquitination and
ultimately degradation by the von Hippel-Lindau (VHL) protein. in hypoxic conditions
however, HIF-1α dimerizes with its beta subunit to activate hypoxia-related elements
including glucose metabolism, cell proliferation, erythropoiesis and apoptosis. It is
difficult to conclude whether an up-regulation or down-regulation of HIF-1α would serve
the human body best. Recent studies have shown that HIF-1α does not serve the
metabolic pathway well, as it promotes glucose intolerance, obesity and insulin
resistance. On the other hand, HIF-1α is beneficial in the vascular pathway, as it
promotes erythropoiesis, which produces erythropoietin that can protect neurons from
degenerating. Although more in-depth studies of HIF-1α are needed, HIF-1α is a good
candidate in treatments for obesity related diseases like chronic liver diseases,
diabetes, and vascular-related diseases such as ischemia, chemotherapy, Parkinson’s
and Alzheimer’s disease.
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Introduction
The liver is a vital organ that functions by regulating hormone production, red-
blood cells production, protein synthesis, as well as regulating oxygen homeostasis and
body metabolism. To maintain oxygen homeostasis, sufficient levels of oxygen need to
be attained at the liver. While oxygen levels of 60-65mmHg is detected in periportal
blood, only 30-35mmHg of oxygen reaches the perivenous sections of the liver
parenchyma (Jungermann et al., 2000). For the reason that liver tissues experience
lower partial pressure of oxygen than the rest of the body does, the liver is significantly
more sensitive to hypoxic conditions. Understanding the hepatic mechanisms of
hypoxia could lead to development of treatments for liver diseases in the future.
There are a series of hypoxia inducible factors (HIFs) that serve as transcription
factors under hypoxic conditions. These hypoxia inducible factors, which include HIF 1,
HIF 2 and HIF 3 have been shown to regulate homeostasis in all cells and tissues. HIFs
are heterodimeric proteins that contain basic helix-loop-helix domains and PAS
domains. The basic helix-loop-helix domains coordinate the dimerization of HIFs so that
DNA binding is possible. Similarly, the PAS domains are also critical to dimerization, as
they are composed of units of amino acids that contain A and B repeats coded by
repeating motifs of His-X-X-Asp (X represents any amino acid). Together, the domains
create a platform for protein-protein interactions that promote dimerization (Semenza et
al., 1997).
HIF 1s have been studied more than the HIF 2s and 3s. The alpha-subunit of
HIF-1 activates hypoxia-regulated elements at the absence of oxygen, while the beta
subunit of HIF-1 focuses on the expression of the HIF-1 transcription factor. Under
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hypoxic conditions, HIF-1α translocates across the nuclear membrane and binds to HIF-
1β in the nucleus, where HIF-1β or the aryl-hydrocarbon-nuclear receptor translocator
[ARNT] is located. Once the active dimerized version of HIF-1 forms, it binds to
hypoxia-responsive elements that trigger signal cascades leading to angiogenesis,
erythropoiesis, pH regulation, apoptosis, cell proliferation, glucose metabolism and
proteolysis (Figure 1) (Buckley et al., 2012). In normoxic conditions however, HIF-1α
remains at low concentrations in the cytoplasm. HIF-1α is targeted by prolyl-
hydroxylases (PH 1, PH 2, PH 3) for immediate hydroxylation. Hydroxylated HIF-1α
then encourages the binding of the von Hippel Lindau tumor suppressor protein (VHL),
leading to the ubiquination and proteasomal degradation of HIF-1α (Figure 2). Proline
hydroxylation does not occur under hypoxic conditions, as hydroxylation requires the
presence of oxygen. If HIF-1α is not hydroxylated, the VHL protein does not bind, and
HIF-1α cannot be degraded (Semenza et al., 1997 and Buckley et al., 2012).
Recently, studies have been focused on the metabolic consequences of hypoxia
and the role of HIF-1α in them. Researchers like Buckley et al. have been working on
finding inhibitors that can disrupt the binding of VHL protein to HIF-1α. As HIF-1α
promotes erythropoiesis, preventing HIF-1α from binding to the VHL protein can lead to
treatments for neurodegenerative diseases.
HIF-1α’s role in metabolic function related to obesity
As a transcription factor, HIF-1α plays a major role in metabolic pathways
especially in cellular hypoxic conditions. Through the activation of HIF-1α by adipose
tissue hypoxia, transgenic mice were shown to have developed mild obesity, resistance
to insulin and glucose intolerance (Halberg et al., 2009). Mice with adipose HIF-1α
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knockouts however, did not develop diet induced obesity or metabolic dysfunction (Jiang
et al., 2011). Since it has been established that diet-induced obesity results in liver
hypoxia (Reinke et al., 2011), further studies were conducted to determine the effects of
HIF-1α inhibition on diet-induced obesity. Recently, diet-induced obesity mice were
treated with HIF-1α anti-sense oligonucleotides (ASO) to examine this possible
pharmacological treatment on the mice’s lipid and glucose metabolism (Shin et al.,
2012). The results were promising, as chronic intraperitoneal injection of HIF-1α ASO
lowered HIF-1α mRNA expression significantly in liver tissue, brown adipose tissue,
epididymal adipose tissue and omental adipose tissue in diet-induced obesity mice. A
decrease in weight of mice was observed after the HIF-1α ASO was administered and
the authors attributed that to the increase in energy expenditure (Shin et al., 2012).
Using methods like the hyperinsulinemic euglycemic clamp, intraperitoneal
glucose tolerance test and insulin tolerance test, the results of Shin et al.’s study
demonstrated a decrease in levels of hepatic glucose output, fasting glucose and
insulin, as well as a decrease in liver lipids and plasma cholesterol levels. These show
that exogenously administered HIF-1α inhibitors have the potential to induce metabolic
benefits in mice with pre-existing diet-induced obesity conditions. As the up-regulation
of HIF-1α leads to hepatic steatosis, HIF-1α ASO treatment could alleviate this condition
by promoting lipid oxidization (Shin et al., 2012).
A similar study by Park et al. also examined the effects of HIF-1α ASO on diet-
induced mice. Although the main focus on the study varied, their results agree with
those produced by Shin et al. in the sense that both reported a decrease in body
weights and fat mass weights. Park et al. proposed that the up-regulation of HIF-1α,
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which promotes angiogenesis, is a key factor of the production of adipocytes in the
development of obesity. Instead of using regular HIF-1α ASO, a mixture of antisense
ASO and low-molecular weight protamine (LMWP) was administered. Park et al.
observed a decrease in subcutaneous adipocyte cell size and a decrease in blood
vessels in the group of ASO-LMWP treated diet-induced obesity (DIO) mice compared
to the untreated DIO group. A decrease in plasma lipid concentrations of the treated
mice also suggests that a HIF-1α inhibitor has the potential to limit angiogenesis and to
reduce hepatic lipogenesis (Park et al., 2012).
Liver cells compensate for hypoxia by increasing HIF-1α
A study by Lau et al. was conducted on rats to determine whether expressions of
HIF-1α vary when the liver undergoes chronic hypoxia. After placing adult male rats in
an acrylic hypoxia chamber at a persistent 10% oxygen for a range of 7 to 28 days,
researchers measured alanine aminotransferase (ALT) and total 8-isoprostane in
serum, as well as measured mRNA expressions of hypoxia related elements like
vascular endothelial growth factor (VEGF), inducible nitric oxide synthase (iNOS),
endothelial nitric oxide synthase (eNOS) and endothelin-1 (ET-1) using RT-PCR.
Hypoxia related elements are signal proteins that increase or decrease in amounts due
to hypoxic conditions. The novel part of this study was the use of electrophoretic
mobility shift assays (EMSA) to detect HIF-1α and NF-kB activity. Transcription factors
like HIF-1α and NF-kB regulate the expression of iNOS and eNOS genes, which is
associated with the regulation of vascular tone. Hypoxia leads to pulmonary
vasoconstriction, so an up regulation of HIF-1α would result in an increase in VEGF,
which produces a vasodilator nitric oxide. The study also showed an up-regulation in
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HIF-1α activity and no difference in levels of ALT, total 8-isoprostane or nitrotyrosine
proteins. Increased HIF-1α activity shows that liver cells have coping mechanisms
when they are subjected to persistent hypoxia. As there was no increase in levels of
liver enzymes or necrosis factors, this indicates no evidence of necrosis and oxidative
stress in rats that were subjected to about 4 weeks of chronic hypoxia (Lau et al., 2013).
The work of Lau et al. contradicts the work of Bianciardi et al., as Bianciardi et al.
reported undetectable levels of HIF-1α expression after their rats were subjected to 2
week of chronic hypoxia treatment (Bianciardi et al., 2006). This could be due to the
difference in HIF-1α expression and activity, or it could be the lower sensitivity in the
latter study’s detection methods. As Lau et al. applied radioisotope EMSA to measure
binding activity of HIF-1α, the authors have claimed that this method yields a much
higher sensitivity and specificity compared to the Western blot method used by
Bianciardi et al (Lau et al., 2013 and Bianciardi et al., 2006). Despite the difference in
methods, the results are inconclusive and further investigation should be conducted to
confirm the findings of each research group.
HIF-1α enhances erythropoietin production
Erythropoietin is a hormone regulated by hypoxia-inducible factors that dictates
red blood cell production. During hypoxic conditions, erythropoietin has been shown to
protect neurons in the brain. Benefits of erythropoietin have been evident in the brain of
stroke patients and animal models, so researchers have been looking into the
neuroprotective capacity of erythropoietin by upregulating erythropoietin (Grimm et al.,
2005). Understanding the mechanisms in which erythropoietin protects neurons in the
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brain could lead to developments of treatments for neurodegenerative diseases in the
future.
Along with HIF-2α, HIF-1α regulates the delivery of oxygen to blood vessels
during hypoxic conditions. Specific roles of HIF-1α and HIF-2α in the induction of
hepatic erythropoietin are still unknown, but the work of Rankin et al. found that HIF-2α
is the dominant factor in the regulation of erythropoietin expression (Rankin et al.,
2007). Results from Rankin et al.’s study also showed that HIF-1 and HIF-2 target the
activation of different genes, as phenotypes from HIF-1 and HIF-2 knockout mice were
compared and distinct differences were noted. HIF-1α and HIF-2α are homologous
proteins, as their % similarity of DNA binding (83.9%) and dimerization (66.5%) are
high. Despite their relatively homologous structure, HIF-1 and HIF-2 have different
functions in regulating erythropoiesis (Rankin et al., 2007).
As the primary function of erythropoietin is to facilitate the production of red blood
cells, erythropoietin was found to be an oxygen-dependent protein in hypoxic brains a
few years ago (Stroka et al., 2001). Recall that erythropoiesis is one of the hypoxia-
related elements in Figure 2, which means that it is one of the downstream effects of the
activation of HIF-1α. Therefore, elevated levels of HIF-1α were found in hypoxic brains
in addition to high levels of erythropoietin. In 1998, Sakanaka et al. published their
findings detailing erythropoietin’s protective role in neurons (Sakanaka et al., 1998).
Subsequently, Marti et al. found that animal stroke models also confirmed that
erythropoietin protects neurons against ischemic brain injury (Marti et al., 2000).
These findings are promising for treatments for neurodegenerative illnesses like
Parkinson’s and Alzhemier’s disease. Because erythropoietin has shown promise in
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protecting neurons, work is being done to focus on mechanisms that can increase levels
of erythropoietin. One of the ways is to upregulate levels of activated HIF-1α so that it
triggers a signal cascade that promotes erythropoiesis (Grimm et al., 2005).
HIF-1α/von Hippel-Lindau protein interactions
Despite the negative effects associated with HIF-1α mentioned previously in this
review article, HIF-1α also activates beneficial hypoxia-related elements like
erythropoietin. The work of Buckley et al. has been performed to disrupt interactions
between HIF-1α and the VHL protein that marks HIF-1α for ubiquitination and ultimately,
degradation. The VHL protein is a portion of a bigger complex that consists of elongin B
and elongin C. As shown in Figure 4, the VHL component is stabilized by right-handed
alpha helices and anti-parallel beta strands. There are two major domains on opposite
ends of the protein. On the N-terminus is the b domain, which is composed of beta
sheets, while the C-terminus houses the smaller a domain, which is composed of alpha
helices (Buckley et al., 2012).
A family of proteins called E3 ubiquitin ligases bind protein targets to mark them
for degradation by proteasomes. These ligases partner with the VHL protein, which is
their substrate recognition subunit, to degrade specific proteins (Buckley et al., 2012).
During normoxic conditions, HIF-1α is one of the proteins that is degradable by the E3
ligase and VHL protein complex (Figure 2). HIF-1α binds to the N-terminus of the VHL
protein, where the beta strands are located, and it is marked for ubiquitination. Since
HIF-1α increases endogenous erythropoietin production when activated, inhibitors have
been designed to attempt to mimic the hypoxia response and disrupt the binding of HIF-
1α to the VHL protein to treat diseases like chronic anemia, ischemia and stroke.
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Figure 3 shows the active site of the VHL protein (light blue protein surface) and HIF-1α
bound to it (Buckley et al., 2012). Due to the highly specific VHL protein, an inhibitor
similar to the structure of HIF-1α is needed to disrupt interactions between the VHL
protein and HIF-1α. After analyzing specific residues on HIF-1α, Buckley et al. found
that Hydroxyproline (Hyp) 564 participates in important interactions with the VHL
protein. Figure 5 shows the location of Hyp 564 at the active site of VHL. That specific
area is crowded, requiring specific twists and turns that only hydroproline resides can fit,
thus a structure was started with the incorporation of hydroxyproline at location 564.
Analogues were then generated using a de novo software BOMB as guidance. The Kd
and IC50 of possible ligands were measured to determine the degree of compatibility
with the VHL active site (Buckley et al., 2012).
Buckley et al. synthesized a compound named 15 that binds at the same site on
the VHL as HIF-1α. Due to the presence of hydroxyproline in the synthesized
compound 15, the ring on the residue accurately mimics interactions that were found in
the protein-protein interface between HIF-1α and the VHL protein. Additionally, the
hydroxyl group also interacted with residues His115 and Ser 111 on the VHL protein,
while the amide group forms interactions with the carbonyl on His 110. Each interaction
that forms between the inhibiting ligand and the VHL protein strengthens the binding
between proteins and decreases the dissociation constant of the ligand-protein
complex. The Kd of 15 was determined to be 5.4uM by isothermal titration colorimetry,
while a nonfluorescent compound that was used as a positive control, managed a Kd of
180nM, indicating tighter binding. This suggests that a lower Kd is needed for 15 to
inhibit the active site of VHL more efficiently.
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Discussion
Under hypoxic conditions, HIF-1α translocates to the nucleus and binds with its
beta counterpart HIF-1β. This newly formed dimer then binds to a series of hypoxia
related elements with some help from co-activators p300 and CBP. P300 and CREB-
binding protein (CBP) aid in the activation of transcription factors to increase gene
expression of hypoxia related elements. Among the many elements triggered are
erythropoiesis, glucose intolerance and insulin resistance (Semenza et al., 1997).
It has been established that an increase in HIF-1α levels in mice lead to the
development of obesity under hypoxic conditions. After hypothesizing that HIF-1α is the
culprit of the development of glucose intolerance, increase in body weight and
development of insulin resistance, Shin et al. found out that HIF-1α ASO can halt the
damaging effects to the metabolic pathways of diet-induced obesity mice under
intermittent hypoxia. Similarly, Lau et al. also saw an increased in HIF-1α activity in rat
models that were persistently exposed to hypoxia. The interesting part of the Lau et al.
study was the claim that liver cells were able to compensate for the hypoxic tissues by
up-regulating HIF-1α. This was shown by the stationary levels of liver enzyme ALT, NF-
kB and total-8 isoprostane, which suggests no evidence of necrosis or oxidative stress
in the hypoxic livers (Lau et al., 2013).
The work of Park et al. and Shin et al. have shown that inhibiting HIF-1α can lead
to weight loss and possibly insulin sensitivity. For HIF-1α inhibitors to be possible
disease treatments, further research has to be done to determine the side effects that
come with down-regulating HIF-1α. Possible side effects include the degeneration of
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neurons, as a decrease in HIF-1α leads to a decrease in levels of erythropoietin (van
Molle et al., 2012).
It has been established that erythropoietin has the ability to protect neurons from
degenerating and thus contribute another possible treatment for neurodegenerative
diseases like Parkinson’s and Alzhemier’s. As a result of this, researchers have been
studying ways to increase erythropoiesis in the brain. One way is to create lead
inhibitors with structures similar to that of HIF-1α (van Molle et al., 2012) or small-
molecule ligands using in silico design co-crystallized with the VHL protein (Buckley et
al., 2012). Using high resolution crystal structures of the VHL protein and HIF-1α,
Buckley et al. were able to scrutinize important protein-protein interactions that need to
be incorporated into the inhibiting ligands. Conserving these important interactions at
the protein-inhibitor interface would promote tighter binding between the protein and the
inhibitor, leading to a more effective competitive inhibition (Buckley et al., 2012).
As a result, the inhibition of HIF-1α binding to the VHL protein would prevent
degradation of HIF-1α even under normoxic conditions. An up-regulation of HIF-1α
would then dimerize with HIF-1β, bind to hypoxia-related elements and promote
erythropoiesis. There is however a significant concern for promoting erythropoiesis, that
is high levels of erythropoietin also promote tumor growth, as erythropoietin stimulates
vessel growth (Ribatti, 2010). Thus, further studies need to be done to propose a
mechanism that promotes erythropoiesis for neuron protection without the side effect of
tumor proliferation.
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HIF-1α can be both beneficial and harmful at extreme concentrations. Since HIF-
1α is a prime candidate in the development of treatment for many diseases, extensive
research needs to be continued to understand this transcription factor.
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Acknowledgements
The author would like to thank Dr. Jennifer Stern, Dr. Sairam Parthasarathy, Dr.
Christopher Sinton and Alexander Bode for their contributions on the discussion of HIF-
1α during lab meetings.
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Figure Legends:
Figure 1: The pathway of HIF-1α in hypoxic conditions. HIF-1α binds to its beta
counterpart in the nucleus then activates hypoxia related elements, which leads to a
signal cascade.
Figure 2: The pathway of HIF-1α in normoxic conditions. HIF-1α is hydroxylated by
prolyl-hydroxylases (PH 1, PH 2, PH3). Then, it binds to the von Hippel-Lindau protein
(VHL) and is targeted for ubiquitination (U). Proteasomes (PS) recognizes the
hydroxylated and ubiquitinated HIF-1α and degrades it. Each pair of red circles
represent molecular oxygen (O2).
Figure 3: HIF-1α at the active site of the von-Hippel Lindau protein. HIF-1α is portrayed
in sticks in this figure with yellow atoms indicating sulfur atoms, blue atoms indicating
nitrogen atoms, red atoms indicating oxygen atoms and green atoms indicating
hydrocarbons. The VHL protein is shown here in cartoon with a light blue surface
transparency of 20% to show the interior of the protein.
Figure 4: Cartoon representation of the VHL protein (blue) with Elongin B (red) and
Elongin C (green). This protein complex is the subunit recognition unit that recognizes
hydroxylated HIF-1α (magenta) and ubiquitinates it for degradation in normoxic
conditions.
Figure 5: HIF-1α at the binding site of the VHL protein (shown here with an all black
surface). All of the atoms on the key amino acid residue Hydroxyproline 564 are labeled
564 to show their positions in the crowded active site.
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Figure 1
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Figure 2
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Figure 3
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Figure 4
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Figure 5