Endothelin-1 induced endoplasmic reticulum stress in disease …jpet.aspetjournals.org/.../06/05/jpet.113.205567.full.pdf · Running Title Page Endothelin-1 induced endoplasmic reticulum

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Title Page

Endothelin-1 induced endoplasmic reticulum stress in disease

Arjun Jain

Swiss National Center of Competence in Research, NCCR TransCure, Switzerland; and Centre

for Trophoblast Research, University of Cambridge, UK

JPET Fast Forward. Published on June 5, 2013 as DOI:10.1124/jpet.113.205567

Copyright 2013 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 5, 2013 as DOI: 10.1124/jpet.113.205567

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Running Title Page

Endothelin-1 induced endoplasmic reticulum stress in disease

Address correspondence to Dr Arjun Jain, 42 Bumplizstrasse, 3027 Bern, Switzerland.

Phone: +41 0786796748; E-mail: [email protected]

Number of text pages: 30

Number of tables: 0

Number of figures: 3

Number of references: 81

Number of words in Abstract: 130

Number of words in body of manuscript: 5867

Abbreviations:

ATF6, activating transcription factor 6; bZIP, basic-leucine zipper motif; CHOP, C/EBP

homologous protein; EAE, experimental autoimmune encephalomyelitis; ECE-1, endothelin-

converting enzyme-1; eIF2α, eukaryotic initiation factor 2α; ER, endoplasmic reticulum; ERAD,

ER-associated degradation; GRP-78 and -94, glucose regulated protein -78 and -94; IP3,

inositol 1,4,5-triphosphate; IRE1α, inositol-requiring 1α; IUGR, intrauterine growth restriction;

MMP-2, matrix metalloprotease-2; mTOR, mammalian target of rapamycin; PERK, double-

stranded RNA-dependent protein kinase (PKR)-like ER kinase; PIP2, phosphatidylinositol 4,5-

bisphosphate; ROS, reactive oxygen species; UPR, unfolded protein response; XBP-1, X-box-

binding protein 1


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Abstract

The accumulation of unfolded proteins in the endoplasmic reticulum (ER) represents a

cellular stress induced by multiple stimuli and pathological conditions. Recent evidence

implicates Endothelin (ET)-1 in the induction of placental ER stress in pregnancy

disorders. ER stress has previously also been implicated in various other disease states,

including neurodegenerative disorders, diabetes and cardiovascular diseases, as has

ET-1 in the pathophysiology of these conditions. However, till date, there has been no

investigation on the link between ET-1 and the induction of ER stress in these disease

states. Based on recent evidence and mechanistic insight on the role of ET-1 in the

induction of placental ER stress, the following review attempts to outline the broader

implications of ET-1 induced ER stress, as well as strategies for therapeutic intervention

based around ET-1.

Body of Manuscript

Endoplasmic Reticulum Stress

The endoplasmic reticulum (ER) is a multifunctional organelle, involved in the synthesis, folding,

and post-translational modification of membrane and secretory proteins. In addition, the ER also

serves as a reservoir of calcium ions (Ca2+) (Zhang and Kaufman, 2008). In the ER lumen, Ca2+

is buffered by calcium-binding proteins. Many of these proteins also serve as molecular

chaperones involved in folding and quality control of ER proteins, and their functional activity

alters with changes in Ca2+ concentration (Michalak et al., 1998). Growth factors, hormones and

stimuli that perturb cellular energy levels, nutrient availability or redox status, all affect ER

calcium storage. Loss of ER Ca2+ homeostasis suppresses post-translational modifications of

proteins in the ER. As a result, misfolded proteins accumulate, provoking ER stress response


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pathways known collectively as the Unfolded Protein Response (UPR) (Brostrom and Brostrom,

2003).

The UPR is induced to restore ER homeostasis. It reduces the burden of new proteins entering

the ER lumen through attenuation of translation, it enhances the protein folding capacity by

increasing ER chaperone proteins (GRP-78 and -94) and folding enzymes, and promotes

degradation of remaining unfolded or misfolded proteins through increased capacity of the

cytosolic ubiquitin-proteasome system (Brostrom and Brostrom, 2003; Yung et al., 2008; Zhang

and Kaufman, 2008). The UPR is initiated by three ER-localized protein sensors; PERK

(double-stranded RNA-dependent protein kinase (PKR)-like ER kinase), IRE1α (inositol-

requiring 1α) and ATF6 (activating transcription factor 6) (Figure 1). In resting cells, all three ER-

stress sensors are maintained in an inactive state through association with the abundant ER

chaperone BiP (immunoglobulin-heavy-chain-binding protein), also referred to as glucose-

regulated protein (GRP)78. During ER stress, GRP78 is sequestered through binding to

unfolded or misfolded polypeptide chains, which leads to the release and consequently

activation of the ER stress sensors (Zhang and Kaufman, 2008).

The release of GRP78 results in activation of PERK through PERK homodimerisation and trans-

phosphorylation. This is the most immediate response of the UPR. Activated PERK then

phosphorylates the eukaryotic initiation factor (eIF2) α subunit, which inhibits non-essential

protein synthesis (Zhang and Kaufman, 2008). This helps promote cell survival by preventing

the influx of more nascent polypeptides into an already saturated ER lumen. The release of

GRP78 also allows IRE1α to dimerize, activating both its protein kinase and endoribonuclease

activity. IRE1α then initiates the removal of a 26-base intron from mRNA encoding X-box-

binding protein 1 (XBP-1), resulting in a translational frameshift and transcription of an XBP-1

isoform with potent activity as a transcription factor, which activates the expression of UPR


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target genes. The third pathway in the UPR is mediated by ATF6. The luminal domain of ATF6

is responsible for sensing unfolded proteins, while the cytoplasmic portion of ATF6 has a DNA-

binding domain containing the basic-leucine zipper motif (bZIP) and a transcriptional activation

domain. In resting cells, GRP78 binds to the luminal domain of ATF6 and hinders the Golgi-

localization signal (Yoshida, 2007). Under conditions of ER stress, the release of GRP78 from

ATF6 allows ATF6 to translocate to the Golgi apparatus, where it is cleaved into an active

cytosolic form. Active ATF6 then migrates to the nucleus and activates the transcription of more

UPR target genes (Zhang and Kaufman, 2008). Essentially, cleaved ATF6 and active XBP-1

function in parallel pathways to induce the transcription of genes encoding ER chaperones and

enzymes that promote protein folding, A final pathway of the UPR is responsible for degradation

of any remaining unfolded or misfolded proteins. The ER-associated degradation (ERAD)

machinery recognizes and translocates these proteins to the cytosol, where they are

ubiquitinated and degraded by the cytosolic proteasome (Yoshida, 2007). Collectively, these

pathways help resolve the protein folding defect and restore homeostasis in the ER. However, if

this fails, apoptosis is then initiated to protect the organism by eliminating the stressed cells that

produce misfolded or malfunctioning proteins (Schroder and Kaufman, 2005). ER-stress

induced apoptosis is mainly mediated by CHOP, a transcription factor that induces the

expression of pro-apoptotic factors and inhibits transcription of anti-apoptotic factors, such as

Bcl-2 (Zhang and Kaufman, 2008).

Malfunction of ER stress responses due to aging, genetic mutations or environmental factors

can result in various diseases, such as diabetes, cardiovascular diseases and

neurodegenerative disorders (Yoshida, 2007; Kim et al., 2008). ER stress has also been

implicated in the pathophysiology of pregnancy disorders (Yung et al., 2008; Burton et al., 2009;

Jain, 2012).


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ER stress in pregnancy disorders; pre-eclampsia and intrauterine growth restriction

Pre-eclampsia and intrauterine growth restriction (IUGR) are major causes of clinical morbidity

and mortality in pregnant women and prenatal infants, affecting between 3-10% of all

pregnancies worldwide (Roberts and Hubel, 1999). A study by Yung et al. (2008) demonstrated

that protein synthesis inhibition and ER stress play a key role in the pathophysiology of both

IUGR and pre-eclampsia (Yung et al., 2008). Increased phosphorylation of eIF2α is found to

inhibit protein synthesis and reduce cell proliferation in pathological placentas. Increased p-

eIF2α levels in the placenta lead to suppression of AKT and proteins/kinases in the mTOR

(mammalian target of rapamycin) pathway (Yung et al., 2008). Reduced activity in this pathway

results in reduced phosphorylation of 4E binding protein 1 (4E-BP1), which is then unable to

bind the translation initiation factor eIF4E and inhibit cap-dependent translation (Hay and

Sonenberg, 2004). ER stress is also proposed to contribute to inflammation in pregnancy

disorders. Following activation of the PERK pathway, there is up-regulation of chaperone

proteins as part of the ATF6 and IRE1α induced stress response. The ER produces ROS

(reactive oxygen species) as a by-product of protein folding, which would be increased under

conditions of ER stress during repeated attempts to refold misfolded proteins. ROS produced

under conditions of ER stress contribute to an inflammatory response. Increased apoptosis in

human term placentas has also been implicated in both pre-eclampsia and IUGR (Ishihara et

al., 2002). As explained above, if the UPR fails to restore ER homeostasis, then apoptosis is

induced, which only occurs under severe conditions of ER stress. Yung et al. (2008) found

increased levels of CHOP in both pre-eclamptic and IUGR placentas compared to normal

controls. A study by Ishihara et al. (2002) also showed elevated apoptosis levels in pre-

eclampsia (Ishihara et al., 2002). Collectively, these studies demonstrate evidence of ER stress

in pregnancy disorders. A recent study by Jain et al. (2012) identified Endothelin (ET)-1 as a key

stimulus of this stress.


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Role of ET-1 in ER Stress

Endothelins are a family of vasoactive peptides that have key physiological functions that

include acting as modulators of the vascular tone, tissue differentiation and cell proliferation

(Nelson et al., 2003). The family of endothelins comprises three isoforms with 21 amino acids

each (ET-1, ET-2, ET-3). ET-1 is the most abundant member of the family of endothelins

(Struck et al., 2005), and is synthesized and secreted by a diverse range of cells. ET-1 exerts its

effects by binding to the endothelin A (ETA) and endothelin B (ETB) receptors, two highly

homologous cell-surface proteins that belong to the G-protein- coupled receptor superfamily

(Karet and Davenport, 1994). Upon binding G-protein- coupled receptors, ET-1 triggers

signaling cascades in a wide variety of target tissues. A recent study by Jain et al. (2012)

demonstrated that ET-1 acting through the ETB receptor activates the PLC/IP3 pathway to

induce Ca2+ release from the ER, which stimulated ER stress in placental tissue. ET-1 induced

an increase in p-eIF2α levels and upregulation of the chaperone proteins, GRP78 and GRP94,

which are all markers of ER stress (Jain et al., 2012). This action of ET-1 on the induction of ER

stress was confirmed by the use of inhibitors acting at different stages of the proposed pathway

(Figure 2).

The study by Jain et al. (2012) also found increased ET-1 levels in both pregnancy disorders,

pre-eclampsia and IUGR, which was in concurrence with previous clinical data (Fiore et al.,

2005). Deficient conversion of maternal spiral arteries (a key underlying feature of pregnancy

disorders) that produces an ischemia/reperfusion-type insult was demonstrated to be a source

of the elevated levels of ET-1. Hence, ET-1 was proposed to be an important stimulus of ER

stress in pregnancy disorders. Many of the molecules that ET-1 was shown to activate have


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also been found to be activated in other tissues and cell types but this was the first report of

their activation by ET-1 in relation to the induction of ER stress. Given the presence and

activation of these molecules in other tissue and indeed other disease states, including

neurodegenerative diseases, broader implications for ET-1 induced ER stress are suggested

over and above the effects in pregnancy.

The following sections describe the evidence for ER stress in different diseases, where ET-1

levels are also increased, and attempt to highlight the likely link between the two, in context of

the recent findings on the induction of placental ER stress by ET-1 in pregnancy disorders.

Neurodegenerative diseases

Unfolded or misfolded proteins that accumulate during conditions of ER stress can form

aggregates in the ER, as well as the cytosol. These aggregates are highly toxic, as they impair

the ubiquitin proteasome pathway (Bence et al., 2001) and excessive accumulation of unfolded

proteins triggers apoptotic pathways. Neurons are thought to be sensitive to protein aggregates

and there are numerous studies that have reported ER stress is involved in neurodegenerative

diseases (Althausen et al., 2001; Milhavet et al., 2002; Hayashi et al., 2005; Hoozemans et al.,

2005; Unterberger et al., 2006; Lin and Popko, 2009; Deslauriers et al., 2011). ET-1 has been

linked to the generation of oxidative damage in neurodegenerative diseases but there has been

no assessment of the role of ET-1 induced ER stress in these conditions.

Multiple Sclerosis

Multiple sclerosis (MS) is characterized by inflammatory demyelination and ensuing

neurodegeneration . ER stress has been proposed to be one mediator of this inflammation, and


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numerous groups have implicated ER stress in MS-related disease mechanisms (Lin and

Popko, 2009; Deslauriers et al., 2011). The study by Deslauriers et al. (2011) found increased

levels of inflammatory cytokines, as well as increased splicing of XBP-1 and higher levels of

GRP78 in the white matter of MS patients compared to healthy controls. ER stress and

inflammation have also been found in the MS model, experimental autoimmune

encephalomyelitis (EAE). EAE is characterized by T cell activation and entry into the CNS with

neuroinflammation, recapitulating many of the events that occur in MS (Kap et al., 2010).

Several markers of ER stress, including GRP78, CHOP and splicing of XBP-1 were all

increased in EAE compared to controls (Deslauriers et al., 2011). The study by Yung et al.

(2008) showed that these same markers of ER stress are also elevated in pregnancy disorders

and the later study by Jain et al. (2012) identified ET-1 as a key stimulus of this stress.

ET-1 is widely distributed in the body, including the neurons and glia in the CNS (Filipovich and

Fleisher-Berkovich, 2008). Furthermore, ET-1 levels are increased in several neurodegenerative

diseases, and thus numerous studies have investigated the role of ET-1 in these conditions.

Speciale et al. (2000) found increased levels of ET-1 and nitric oxide (NO) in the cerebrospinal

fluid of patients with MS (Speciale et al., 2000). Other studies have also demonstrated

increased ET-1 plasma levels in MS patients compared to sex- and age- matched healthy

controls (Haufschild et al., 2001; Pache et al., 2003).

Activation of glial cells in the brain is believed to contribute to the pathogenesis of multiple

sclerosis (Matsumoto et al., 1992). Activation of glia leads to production of proinflammatory and

cytotoxic factors, such as prostaglandins and NO that have been connected to increased

neurotoxicity in in vitro neuron-glia cultures. ET-1 has been proposed to significantly enhance

the synthesis of prostaglandin E2 and NO in glial cells (Filipovich and Fleisher-Berkovich, 2008).

Although the exact mechanism by which ET-1 stimulates glial prostaglandin E2 synthesis is not


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clear, one possibility is activation of PLC, which participates in a secondary pathway of

arachidonic acid release, the first step in prostaglandin E2 synthesis. The regulation of NO

synthesis by ET-1 is also thought to be via the stimulation of PLC. ET-1 mediated activation of

PLC leads to the formation of IP3 that stimulates Ca2+ release from the ER. The resulting rise in

cellular Ca2+ can induce the production of NO (Filipovich and Fleisher-Berkovich, 2008). The

study by Jain et al. (2012) demonstrated that ET-1 induces ER stress via activation of the

PLC/IP3 pathway to induce Ca2+ release from the ER, which disrupts ER Ca2+ homeostasis and

hence stimulates ER stress. As the upregulation of pro-inflammatory factors (prostaglandins and

NO) is considered to be mediated via PLC activation, there is considerable overlap with the

signaling pathways implicated in the ET-1 induced ER stress response. Therefore, it is likely

that in addition to the effects described above, the elevated levels of ET-1 in multiple sclerosis

also contribute to inflammatory demyelination via induction of ER stress.

Activation of glial cells in the brain is also thought to contribute to the pathogenesis of

Alzheimer’s disease (Rogers et al., 1988).

Alzheimer’s disease

Alzheimer’s disease is a progressive neurodegenerative condition, characterized by

extracellular lesions in the cerebral cortex composed largely of aggregates of amyloid-β peptide

(Gething, 2000). Mutations in the presenilin-1 and presenilin-2 proteins have been proposed to

cause early-onset Alzheimer’s disease by increasing the generation and secretion of amyloid-β

peptides (Aβ) in the brain. Aβ has been reported to induce ER stress leading to neuronal cell

death (Costa et al., 2012). Recent studies have found that 4-phenylbutyrate attenuates the

activation of ER stress and associated neuronal cell death, by activating the HRD1 ubiquitin

ligase that promotes Aβ clearance (Kaneko, 2012; Mimori et al., 2012). In addition to their


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effects on Aβ generation, mutant versions of presenilin-1 have been found to induce altered ER

Ca2+ homeostasis and render cultured neurons more susceptible to cell death induced by ER

stress (Terro et al., 2002). Indeed, a study by Milhavet et al. (2002) found that brains of mice

harboring Alzheimer’s disease-associated mutants of presenilin-1 have increased levels of

CHOP, a marker of ER stress-induced cell death (Milhavet et al., 2002).

Autopsy studies have also shown that the PERK-eIF2α pathway is hyperactive in the brains of

patients with Alzheimer’s disease (Unterberger et al., 2006), further implicating ER stress in the

pathogenesis of Alzheimer’s. In addition, a study by Hoozemans et al. (2005) found increased

expression of the ER chaperone GRP78 (also indicative of UPR activation) in cases of

Alzheimer’s disease compared with healthy controls (Hoozemans et al., 2005). ET-1 has

previously been shown to activate the PERK-eIF2α pathway and also induce GRP78 levels in

pregnancy disorders (Jain et al., 2012). Given that ET-1 levels are elevated in Alzheimer’s

disease (Palmer et al., 2009; Palmer et al., 2012), ET-1 might compound the effect of presenilin-

1 on disrupting ER Ca2+ homeostasis and rendering neurons more susceptible to cell death

induced by ER stress. Previous research has looked at the vasoactive function of ET-1 as a

pathological factor responsible for the decrease in cerebral blood flow in Alzheimer’s disease

(Palmer et al., 2009). Based on the evidence at hand, further research is required to investigate

the role of ET-1 in inducing ER stress in Alzheimer’s disease.

Acute Neurodegeneration

Apart from the more chronic neurodegenerative diseases, ER stress is also shown to be present

in acute brain disorders, such as ischemia. Focal cerebral ischemia in mice has been shown to

upregulate phospho-eIF2α levels (Althausen et al., 2001), while global ischemia in mice has

been found to induce CHOP and ATF-4 (Hayashi et al., 2005). It has been reported that


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hippocampal neurons from CHOP-deficient mice are more resistant to cell death induced by

ischemia/reperfusion compared with normoxic-controls (Tajiri et al., 2004). Fewer neurons

degenerated in the CHOP-/- mice after ischemia, suggesting an important role for ER stress in

ischemia/stroke. Ischemia/reperfusion has been demonstrated to also be a potent stimulus for

induction of ET-1 levels (Jain et al., 2012) and given the prevalence of elevated ET-1 levels in

neurodegenerative diseases and the concomitant induction of ER stress, ET-1 is likely one

causative factor of this stress.

Diabetes

Diabetes mellitus (DM) is a metabolic disorder characterized by varying or persistent

hyperglycemia due to reduced insulin action. Type 1 diabetes is characterized by a severe lack

of insulin production due to specific destruction of the pancreatic β-cells that typically develops

over several years (Eizirik et al., 2008). Type 2 diabetes results from β-cell dysfunction with a

reduced ability of the pancreatic β-cells to secrete enough insulin to stimulate glucose utilization

by peripheral tissues (Mathis et al., 2001).

Accumulating evidence suggests that reduction in β-cell mass, due to increased β-cell apoptosis

and defective β-cell regeneration, is a key component in both types of diabetes (Mathis et al.,

2001). Studies indicate a role of the ER in sensing and transduction of apoptotic signals. One of

the first evidence of the involvement of ER stress in diabetes came from studies on the Akita

mouse, which is a spontaneously diabetic model, characterized by progressive hyperglycemia

with reduced β-cell mass. Genetic analyses revealed that a mutation in the insulin 2 gene

(Cys96Tyr) is responsible for the diabetic phenotype in this mouse (Wang et al., 1999). Further

investigations have revealed that progressive hyperglycemia in the mouse was accompanied by

elevated levels of ER stress markers, such as GRP78 and CHOP. Since proinsulin is a major


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ER client protein in pancreatic β-cells, it was suggested that its malfolding is the cause of this

ER stress (Sundar Rajan et al., 2007).

Further evidence implicating ER stress in the pathophysiology of diabetes came from studies on

Wolcott-Rallison syndrome (WRS) (Delepine et al., 2000). WRS is a rare, autosomal recessive

disorder characterized by early infancy onset diabetes mellitus. Mutations in the EIF2AK3 gene,

which codes for pancreatic PERK, were found to be the underlying cause of this disorder. In

addition, Harding et al. (2000) found that Perk -/- mice developed a clinical syndrome similar to

that seen in WRS patients. Perk -/- cells were unable to phosphorylate eIF2α and attenuate

translation in response to ER stress. The absence of PERK rendered these cultured cells prone

to apoptosis that lead to progressive destruction of β-cells (Harding et al., 2000).

Nitric oxide, one of the important effectors of β-cell death in type 1 diabetes and vascular

complications in type 2 diabetes, has been shown to exert its effects by triggering ER stress

(Oyadomari et al., 2001). Oyadomari et al. (2001) have shown that NO depletes ER Ca2+, which

induces ER stress and leads to apoptosis. NO-depletion of ER Ca2+ is thought to occur either by

inhibition of Ca2+ uptake from cytosol through SERCA (sarco/endoplasmic reticulum ATPase) or

by activation of Ca2+ release to the cytosol through RyR. ET-1 is a potent stimulus of NO

(Stricklett et al., 2006), and given the recent evidence implicating ET-1 in the induction of ER

stress by stimulating Ca2+ release through the IP3 receptor (Jain et al., 2012), ET-1 could be a

potent stimulus of ER stress in diabetes through this dual action culminating in disruption of ER

Ca2+ homeostasis. This is consistent with the finding by several groups that ET-1 levels are

increased in diabetes compared to control subjects (Seligman et al., 2000; Schneider et al.,

2002). Many of the markers of ER stress implicated in diabetes that are discussed above have

also been shown to be activated by ET-1. The study by Jain et al. (2012) demonstrated that ET-

1 induces increased levels of the ER chaperone protein GRP78 and also directly affected the

PERK pathway. It was also proposed that in vivo, ET-1 may act in synergy with other


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pathological factors (NO in diabetes?) to induce more potent ER stress, with the activation of

additional pro-inflammatory pathways and induction of apoptosis. The sustained elevated

plasma levels of ET-1 found in diabetes could act continually on pancreatic β-cells to further

exacerbate this effect.

Cardiovascular disease

ER stress is also implicated in cardiovascular diseases such as cardiac hypertrophy, heart

failure, atherosclerosis, and ischemic heart disease.

Cardiac hypertrophy and heart failure

Cardiac hypertrophy is an adaptive response of the heart to hemodynamic overload, during

which terminally differentiated cardiomyocytes increase in size without undergoing cell division.

Although initially this hypertrophic response may serve to compensate cardiac function,

prolonged hypertrophy can become detrimental, resulting in cardiac dysfunction and heart

failure (Wang et al., 2003).

Morphological analysis of degenerated cardiac muscle cells in patients with cardiac hypertrophy

revealed dilated ER cisternae, which is an indicator of a stressed ER (Maron et al., 1975). Yung

et al. (2008) also demonstrated this morphological change in the ER cisternae that

accompanied elevated levels of ER stress markers in pregnancy disorders, such as pre-

eclampsia and IUGR (Yung et al., 2008). The later study by Jain et al. (2012) then

demonstrated ET-1 as an important stimulus of this ER stress. Plasma ET-1 levels are found to

be elevated in cases of heart failure (von Lueder et al., 2004).

Several studies have found a marked increase in GRP78 expression in hypertrophic and failing

hearts, suggesting that activation of the UPR is associated with pathophysiology of heart failure


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in humans (Okada et al., 2004; Dally et al., 2009). Further studies in a mouse model

demonstrated that prolonged ER stress induced CHOP-mediated apoptosis in heart failure but

not hypertrophic hearts, and it was suggested that the CHOP-dependent cell death pathway

may be involved in the transition from cardiac hypertrophy to heart failure (Minamino and

Kitakaze, 2010). The role of ET-1 induced ER stress in cardiomyopathy is supported by a recent

study that found querticin treatment gave a decrease in myocardial levels of ER stress,

accompanied by a significant reduction in ET-1 levels (Arumugam et al., 2012).

Atherosclerosis

Atherosclerosis is a disease wherein arteries harden and narrow because of the accumulation

of plaque (made up of fatty substances, cholesterol, calcium and cellular waste) in the arterial

inner lining, leading to heart attack or stroke (Yoshida, 2007). Accumulation of homocysteine

has been found to be a risk factor for atherosclerosis (Lawrence de Koning et al., 2003).

Homocysteine was found to induce ER stress as evinced by increased expression of GRP78,

GRP94 and CHOP (Werstuck et al., 2001). The induction of CHOP promotes macrophage

apoptosis, which leads to an accumulation of macrophage debris that form plaques in blood

vessels. These then contribute to atherosclerosis observed in hyperhomocysteinemia (Yoshida,

2007). Consistent with this, CHOP-/- macrophages are less sensitive to apoptosis induced by

homocysteine. These findings strongly support a role for ER stress-induced apoptosis in the

development of atherosclerosis. However, there has been no investigation into the mechanism

of induction of this ER stress by homocysteine. Interestingly, homocysteine has been found to

induce ET-1 levels in bovine aortic endothelial cells (Sethi et al., 2006). Indeed, ET-1 levels are

elevated in atherosclerotic plaques from human tissue (Zeiher et al., 1995) and these elevated

levels of ET-1 could be one source of ER stress observed in atherosclerosis.


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Ischemic heart disease

Atherosclerosis is causally involved in ischemic diseases, such as myocardial infarction and

cerebral infarction (Hansson, 2005; Myoishi et al., 2007). Ischemic heart disease is a condition

characterized by reduced blood supply to the heart. In conditions of cerebral infarction and

acute myocardial infarction, the most effective therapy is early and successful reperfusion.

However, recanalization of an occluded artery can cause damage through ischemia/reperfusion

injury (Miyazaki et al., 2011). A natural cause of reperfusion injury is coronary artery vasospasm

followed by coronary artery dilation (Hoffman et al., 2004). Ischemia/reperfusion induced

generation of ROS and depletion of ATP reduce Ca2+ storage within the ER, as ROS can

damage the endoplasmic reticulum Ca2+-uptake system through oxidation of essential thiol

groups on the transmembrane channels, while low ATP results in inhibition of ATP-dependent

sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (Yung et al., 2008). The resulting loss of

Ca2+ homeostasis, compounded by the low ATP concentrations, leads to suppression of ATP

and Ca2+-dependent post-translational modifications (Brostrom and Brostrom, 2003). As a

result, misfolded proteins accumulate that then provoke ER stress response pathways. Azfer et

al. (2006) reported that ER stress-associated genes, such as CHOP are induced in the heart of

an ischemic heart disease mouse model (Azfer et al., 2006). Other studies also found increased

ER stress markers in cardiomyocytes from near the site of myocardial infarction in mice as well

as humans (Thuerauf et al., 2006; Severino et al., 2007).

As discussed above, the study by Jain et al. (2012) demonstrated that ischemia/reperfusion is

also a potent stimulus of ET-1. These findings were consistent with previous work that found

ET-1 levels are stimulated under oxidative stress; ROS that have been implicated in the

pathophysiology of renal ischemia/reperfusion injury stimulate ET-1 production (Hughes et al.,

1996). Incubation of human mesangial cells with ROS donors, such as xanthine/xanthine

oxidase and hydrogen peroxide caused a dose-dependent increase in ET-I levels. Therefore,


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ET-1 levels stimulated under ischemia/reperfusion may compound the effect of ROS and low

ATP-levels during ischemia/reperfusion on induction of ER stress.

Collectively, ET-1 induced ER stress is likely an important pathological factor in the diseases

discussed. The following sections describe potential strategies for therapeutic intervention to

block pathology associated with potential ET-1 induced ER stress in disease.

Receptor antagonists as potential therapeutic tools against ET-1 induced ER stress

The study by Jain et al. (2012) demonstrated that ET-1 induces ER stress via the ETB receptor-

mediated activation of the PLC/IP3 pathway that leads to a disruption in ER Ca2+ homeostasis

(Jain et al., 2012). The action of ET-1 via the ETB receptor was confirmed using an ETB

receptor antagonist, BQ788 (N-cis-2,6-dimethylpiperidinocarbonyl-L--methylleucyl-D-1-

methoxycarbonyltryptophanyl-D-norleucine), which is a potent and selective inhibitor of ET-1

binding to the ETB receptor. This compound therefore provides a useful tool to block ET-1

induced ER stress via the ETB receptor.

As explained above, release of inflammatory mediators by glial cells is thought to play an

important role in neuroinflammation that contributes to the pathogenesis of neurodegenerative

diseases, such as MS and Alzheimer’s disease (Rogers et al., 1988; Filipovich and Fleisher-

Berkovich, 2008). BQ788 has been found to inhibit the production of both NO and prostaglandin

E2 in glial cell cultures (Filipovich and Fleisher-Berkovich, 2008). Accordingly, the study by

Filipovich et al. (2008) proposes that BQ788 may have therapeutic potential in pathological

conditions of the brain

ET-receptor antagonists have also been investigated in a variety of cardiovascular conditions,

including hypertension, atherosclerosis and heart failure. In animal models of chronic heart

failure, prolonged ETA receptor blockade improves cardiac function and prolongs survival


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(Luscher and Barton, 2000). Treatment of heart failure patients with the dual ET-receptor

antagonist, bosentan, has also been found to help lower pulmonary artery pressure (Sutsch et

al., 1998). Dual ETA/ETB receptor blockade by bosentan also yields improvement in certain

patients with coronary atherosclerosis (Wenzel et al., 1998). Given this enhanced effect of dual-

antagonism of the ET-receptors indicates that in addition to blocking the vasoconstrictive effects

of ET-1 via the ETA receptor, blocking the ETB receptor may have the additional advantage of

blocking the pro-inflammatory effects of ET-1, including the potential induction of ER stress.

Further studies will indicate the precise contribution of ETB receptor blockade on ER stress

induced pathology.

Dual ET receptor antagonists have also been applied in diabetes. A study by Hocher et al.

(2001) found that dual ETA/ETB receptor antagonists reduce proteinuria and normalize renal

matrix protein expression in hyperglycemic rats with streptozotocin-induced diabetes (Hocher et

al., 2001). Blockade of the ETB receptor was proposed to reduce glomerular NO synthesis. As

discussed above, NO is an important effector of β-cell death in type 1 diabetes and vascular

complications in type 2 diabetes, through its effects on ER stress (Oyadomari et al., 2001).

Therefore, ETB receptor antagonism might be useful for the dual blockade of ET-1 induced NO,

as well as the direct induction of ER stress by ET-1.

Although, the use of ET receptor antagonists has been useful in ameliorating disease pathology,

there are certain drawbacks to consider. The use of ET antagonists has previously been

associated with side-effects, such as an increase in heart rate, facial flush, and/or facial edema,

(Fleisch et al., 2000) Also, certain ET antagonists may interfere with anticoagulants, such as

warfarin, and therefore pose a risk of thrombosis (Weber et al., 1999). These studies indicate

the importance of only regulated antagonism of the ET receptor system as a therapeutic tool. An


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alternative strategy for prevention of ET-1 induced ER stress would be to target the actual

elevated levels of ET-1 in pathological conditions.

Targeting the elevated levels of ET-1 in pathological conditions

ET-1 is derived from a larger precursor peptide (pre-proET-1; 212 amino acids) that is first

cleaved at specific basic residues to give rise to bigET-1 (38 amino acids), and subsequently

cleaved to generate mature ET-1 (Struck et al., 2005). The latter is achieved by the action of the

endothelin-converting enzyme (ECE-1), which cleaves at the motif Trp73↓Val74.

Given the role of ECE-1 in ET-1 production, inhibition of this enzyme might provide a useful

strategy to lower the pathological increase in ET-1 levels in disease. Accordingly, a study by

Minamino et al. (1997) found that ECE-1 may contribute to the process of injury-induced

neointimal formation and atherosclerosis through increased production of ET-1, which could be

blocked using the ECE-1 inhibitor, phosphoamidon. Similarly, blocking the elevated ECE-1

expression levels in failing human hearts using ACE inhibitor therapy helps reduce elevated

plasma ET-1 levels to normal values, which resulted in additional arterial vasodilatation,

improved cardiac index, and decreased systemic and pulmonary vascular resistance (Galatius-

Jensen et al., 1996).

While there are numerous studies on ECE regulation of the ET system in cardiovascular

diseases, and indeed investigation into inhibitors for therapeutic intervention, there is only very

limited data on ECE antagonists in neurodegenerative diseases. However, previous research

has found increased ECE activity in neurodegenerative conditions. It is known from animal

models that matrix metalloprotease-2 (MMP-2) is identical to ECE, in that it cleaves bigET-1 to

the mature active ET-1. MMP-2 is expressed in and around MS plaques (Leppert et al., 2001)

and it has been suggested that MMP-2 is at least one cause of the elevated levels of ET-1 in the


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cerebrospinal fluid and plasma of patients with MS (Speciale et al., 2000; Pache et al., 2003).

Another study found increased levels of ECE-2 in Alzheimer’s disease (Palmer et al., 2009).

Neural tissues are abundant in ECE-2, which is an isoform of ECE-1 with 59% sequence

homology and similar catalytic activity (Palmer et al., 2009). Palmer et al. (2009) propose that

increased ECE-2 levels in Alzheimer’s disease are a source of the increased ET-1 and reduced

cerebral blood flow prevalent in Alzheimer’s disease. Given the evidence of increased ECE

activity in neurodegenerative diseases, further research is warranted to investigate the precise

therapeutic value of ECE antagonism.

Studies have also found increased ECE-1 expression and activity in diabetes (Anstadt et al.,

2002). Dual inhibition of neutral endopeptidase and ECE by SLV306 reduces proteinuria and

urinary albumin excretion (improves kidney function) in diabetic rats (Thone-Reinke et al.,

2004).

However, although inhibition of ECE blocks the production of ET-1, recent identification of ECE-

independent pathways contributing to ET-1 formation, such as non-ECE metalloproteases (in

multiple sclerosis) (Leppert et al., 2001) and the renin-angiotensin system (in cardiovascular

diseases) (d'Uscio et al., 1998), might limit the effectiveness of these drugs. The author would

like to propose an alternate, novel strategy for targeting the elevated ET-1 levels in pathological

conditions as a means of blocking the ET-1 induced ER stress; using ET-1 traps.

Previous research has investigated the use of cytokine traps to target the elevated levels of

specific cytokines in disease states as a means of blocking their pathological activity.

Economides et al. (2003) constructed cytokine traps by engineering inline fusions of the

extracellular domains of target cytokine receptors (e.g. IL-6Rα and gp130). These were fused

to the Fc portion of human IgG1, which directs formation of disulphide-linked dimers, so that

transfection of expression constructs into mammalian cells results in secretion of the desired


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dimeric inline cytokine trap (Figure 3). Such soluble receptors bind cytokines with high affinity

and were found to be potent cytokine blockers. The use of these traps was verified in both in

vitro and in vivo models, where they were found to potently block target cytokine action and also

displayed good bioavailability in vivo. The same strategy was applied for other cytokines,

including IL-1 and IL-4.

Given that the structure of human ET-1 has been elucidated, including characterization of the

receptor binding site, development of ET-1 traps might provide a useful strategy for therapeutic

intervention in the diseases described above. The three-dimensional structure of ET-1 has been

determined using X-ray crystallography (Janes et al., 1994). The overall structure of the

molecule consists of an N-terminal extended beta-strand with a bulge between residues 5 and

7, followed by a central region that consists of a hydrogen-bonded loop between residues 7 and

11. The C-terminal is a single, long, somewhat irregular helix that extends to the end of the

molecule. The overall tertiary structure of the (N-terminal) head group region, as well as the

nature of the side chain at position 2 appears to be important for specificity of binding of the

ETA receptor, while the ETB receptor binding pocket primarily recognizes determinants that lie

in the helical (C-terminal) tail of ET. The crystal structure of ET-1 would be useful for aiding our

understanding of the nature of the receptor/ligand binding sites and for providing a basis for the

design of ET-1 traps. Key differences in the structure of ET-1 distinguish it from its analogues

and must be considered in the design of such traps, which would need to be specific for ET-1.

For example, a Ser/Thr replacement at position 2 in ET-3 results in a considerable drop in

binding affinity for the ETA receptor. The longer Thr side chain is found to not fit as well into a

complementary binding pocket of the ETA receptor.

As described above for cytokines, ET-1 traps can be designed that bind the ET-1 receptor-

binding domain and hence render the targeted ET-1 inactive. Following clinical assessment of

the extent of elevated ET-1 plasma levels in a patient, one could modulate the dose of ET-1


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traps to be administered in order to target only the ‘excess’ levels of ET-1. In a way, the ET-1

traps would ‘sponge’ up the extra, pathological levels of ET-1 without disrupting the

physiological activities of ET-1. Further investigation into the design and engineering of such

traps would allow an evaluation of the potential application of ET-1 traps for therapeutic

intervention.

Conclusions

The ubiquitous distribution of ET-1 and its receptors implicates its involvement in a wide variety

of physiological and pathological processes in the body. This review highlights the likely link

between increased ET-1 levels in various pathological conditions and the prevalence of ER

stress in these same diseases. Indeed, both ET-1 and ER stress are also implicated in several

other pathological conditions not addressed in this review, including different cancers and

HIV/AIDS. It is hoped that the links outlined in this review will prompt future research into the

precise mechanism of action of ET-1 in inducing ER stress in each individual disease and

subsequent investigation into potential therapeutic strategies for circumventing this stress,

including those outlined in this review. A fuller investigation into this may lead to the

development of therapies for one or more of the devastating diseases implicated.

Acknowledgements

The author would like to thank his family for all their love and support; special thanks to Ashok

and Kirti Jain, T3LOs, Ira, Santosh Kumar Jain, Leela Jain, Shivaling Hiremath and Prema

Hiremath

Authorship contribution

Participated in research design: NA

Conducted experiments: NA


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Contributed new reagents or analytic tools: NA

Performed data analysis: NA

Wrote or contributed to the writing of the manuscript: Jain

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Figure Legends

Figure 1. Mammalian UPR pathways. Accumulation of misfolded or unfolded proteins in the ER causes

ER stress, which induces the UPR. PERK, IRE1α and ATF6 are released from BiP/GRP78, promoting the

induction of downstream signaling pathways in an attempt to restore ER homeostasis (reproduced from

Zhang and Kaufman (2008) (Zhang and Kaufman, 2008)).

Figure 2. Potential mechanism by which endothelin-1 (ET-1) induces ER stress in pathological

pregnancies. ET-1 binding the ETB receptor (ETBR) activates phospholipase-C (PLC)-catalysed

hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to produce diacylglycerol (DAG) and inositol

1,4,5-triphosphate (IP3). IP3binding the IP3 receptor (IP3R) stimulates Ca2+ release from the ER, which

induces ER stress. BQ788, an ETB receptor antagonist, U73122 (1-(6-((17_-3-methoxyestr-1,3,5(10)-


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trien-17-yl)amino)-hexyl)-1H-pyrrole-2,5-dione), an inhibitor of PLC activation and Xestospongin-c, an IP3

receptor inhibitor, all block the induction of ER stress by ET-1 (adapted from Jain et al. (2012)).

Figure 3. Cytokine traps. Extracellular domains of cytokine receptors arranged inline and then fused to Fc

portion of human IgG1 (adapted from Economides et al. (2003)).


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