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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.
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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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