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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: ccheah@email.arizona.edu

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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