Download Signal Transduction From the Endoplasmic Reticulum to the Cell

PHYSIOLOGICAL REVIEWS
Vol. 79, No. 3, July 1999
Printed in U.S.A.
Signal Transduction From the Endoplasmic Reticulum
to the Cell Nucleus
HEIKE L. PAHL
Department of Experimental Anesthesiology, University Hospital Freiburg, Freiburg, Germany
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Pahl, Heike L. Signal Transduction From the Endoplasmic Reticulum to the Cell Nucleus. Physiol. Rev. 79: 683–701,
1999.—The endoplasmic reticulum (ER) serves several important functions. Cholesterol, an essential component of
cellular membranes, is synthesized on the ER surface. Inside the organelle, proteins destined for secretion or
transport to the cell surface are folded and become glycosylated. Because these processes are essential for cell
viability, a disturbance in ER function presents significant stress to the cell. In response to ER stress, three distinct
signal transduction pathways can be activated. Two of these, the unfolded protein response and the ER-overload
response, respond to disturbances in protein processing. The third, the sterol regulatory cascade, is activated by
depletion of cholesterol. This review summarizes the recent advances in our understanding of these ER-nuclear
signal transduction pathways. In addition, it points to novel regulatory mechanisms discovered in these pathways,
which may be widely used in other systems.
I. INTRODUCTION
The endoplasmic reticulum (ER) is one of the largest
cell organelles, its membranes constituting over one-half
of the total membranes in a cell. The ER lumen, the
internal space, comprises over 10% of the cell volume.
This vast structure has two essential functions. 1) Proteins destined for transport to other organelles, secretion,
or expression on the cell surface are synthesized on the
ER surface. During translation, they are translocated into
the ER lumen through a pore in the ER membrane. Inside
the organelle, they are folded, sometimes with the aid of
chaperone proteins, and become glycosylated. A quality
control mechanism ensures that only correctly folded
proteins exit the ER. Incorrectly folded proteins are retained and ultimately degraded. 2) Synthesis of lipids and
cholesterol takes place on the cytoplasmic side of the ER
membrane (70). Therefore, the ER is the production site
for all components of cellular membranes, proteins, lipids, and sterols.
Given the importance of this organelle, its proper
function is essential to the cell. Therefore, it seems only
0031-9333/99 $15.00 Copyright © 1999 the American Physiological Society
natural that mechanisms have evolved to signal a disturbance in ER function. Various conditions can interfere with ER function, and for the purpose of this
review, these are collectively called ER stress. Endoplasmic reticulum stress can arise from a disturbance
in protein folding, leading to an accumulation of un- or
misfolded proteins in the organelle. Starvation of glucose also leads to protein accumulation in the ER, since
glycosylation cannot proceed at the required rate. Likewise, the ER can become saturated when too many
proteins are produced that need to be processed by the
organelle. Finally, starvation of cholesterol, an essential component of cellular membranes, also elicits ER
stress.
Cells respond to most stress situations by changing
protein production; synthesis of those proteins that may
help to alleviate the stress is increased (9). This upregulation occurs mainly at the level of transcription so that
more mRNA molecules encoding the required proteins
are produced. Increased transcription is in turn achieved
by activating transcription factors, proteins which bind
specific DNA sequences in the promoter or enhancer
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I. Introduction
II. Signaling From the Endoplasmic Reticulum to the Nucleus
A. The unfolded protein response
B. A novel role for transcription factor NFkB
C. The ER-overload response
D. ER-nuclear signaling by SREBP
III. Summary
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HEIKE L. PAHL
1. Conditions that induce the unfolded protein
response
TABLE
Drugs perturbing ER function
Tunicamycin
2-Deoxyglucose
Brefeldin A
Castanospermine
Glucosamine
Thapsigargin
AlF42
Glucose starvation
Reducing agents
2-Mercaptoethanol
Dithiothreitol
Heavy metals
Cobalt
Nickel
Overexpression of mutant proteins that cannot fold correctly
Calcium ionophores
A-23187
Ionomycin
ER, endoplasmic reticulum.
It was subsequently shown that an accumulation of correctly folded proteins, which cannot be processed, such
as the immunoglobulin heavy chain in the absence of light
chain, also induces the UPR. Yeast cells contain proteins
homologous to the mammalian GRP and can also mount
an UPR (82). Because of the elegant genetic experiments
that can be performed in yeast, more is known about the
UPR in this system than about its mammalian counterpart
(90). Therefore, the Saccharomyces cerevisiae UPR is
discussed first. Because it is not yet clear whether individual elements of the pathways are evolutionarily conserved, the mammalian UPR is described separately.
1. The UPR in yeast
II. SIGNALING FROM THE ENDOPLASMIC
RETICULUM TO THE NUCLEUS
A. The Unfolded Protein Response
Kozutsumi et al. (38) were the first to propose a
signal transduction pathway that is activated by an ER
stress signal. This group showed that the expression of a
mutant influenza hemagglutinin, which does not fold correctly, but not of the wild-type protein, induced the expression of genes for several ER-resident proteins. These
proteins, first named glucose-regulated proteins (GRP)
because of their induction by glucose starvation (40),
include the heavy-chain binding protein BiP/GRP78,
GRP94, protein-disulfate isomerase (PDI/ERp59), and
ERp72. The GRP facilitate protein folding in the ER. In
this way, they reduce the number of misfolded proteins
and thus alleviate ER stress. Because a common stimulus
for the induction of GRP is the presence of misfolded
proteins in the ER (38), this pathway was named the UPR.
Yeast cells can be treated with a variety of agents that
perturb ER function, for example, glycosylation inhibitors
such as 2-deoxyglucose or reducing agents such as 2-mercaptoethanol, which prevents disulfide bond formation
(see Table 1). All these treatments induce the transcription of a group of ER-resident proteins (82, 90). These
include a member of the 70-kDa heat shock protein family
called KAR2, a peptidyl-prolyl cis-trans isomerase (PPIase) encoded by the FKB2 gene, a protein disulfide
isomerase-like protein named EUG1, and the protein disulfide isomerase PDI1. These proteins function to facilitate protein folding. Therefore, their increased expression, induced by the presence of unfolded proteins in the
ER, should reduce stress to the organelle.
How does the presence of misfolded proteins in the
ER activate transcription in the cell nucleus? Deletion
analysis of the KAR2 promoter identified a 22-bp element,
the UPR element (UPRE), which is conserved in the
promoters of all UPR target genes and is required for their
induction by ER stress (60). Moreover, the UPRE confers
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regions of genes. Their binding increases the rate of transcription of this gene.
Until recently, however, very little was known about
how cells respond to ER stress, which transcription factors are activated, and by what mechanism they are induced. This review focuses on three different types of ER
stress: 1) the presence of mis- or unfolded proteins in the
organelle, 2) the overloading of the ER with correctly
folded proteins, and 3) the starvation of cholesterol. Each
stress situation triggers a unique cellular response, using
various signal transduction pathways to induce specific
transcription factors.
The first of these pathways to be described, the unfolded protein response (UPR), has been known for 10
years. It is activated, as the name suggests, by the presence of mis- or unfolded proteins in the ER. Of the three
pathways, it is the only one conserved, at least in part,
between humans and yeast. The more recently discovered
sterol regulatory element binding protein (SREBP) pathway regulates cholesterol synthesis in the ER membrane
in response to sterol plasma levels. In both of these
pathways, novel mechanisms of transcription factor activation have been described, which may prove to be paradigms for processes widely used in other pathways. The
most recently described ER-nuclear signal transduction
pathway, ER-overload response (EOR), a stress triggered
by congestion of the organelle with too many proteins,
activates a well-characterized transcription factor, nuclear factor kB (NFkB). However, it uses a novel pathway
in the process.
This review compares and contrasts these signal
transduction pathways, pointing out questions that need
to be addressed in the future.
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ENDOPLASMIC RETICULUM SIGNAL TRANSDUCTION
685
ER stress inducibility on a heterologous promoter. Mori et
al. (57) recently conducted a detailed mutational mapping
of the UPRE. They identified an E-box-like palindrome,
separated by one cytosine residue (59-CAG C GTG-39),
which is essential for UPR function. All five yeast UPR
target genes (see Fig. 1) contain a functional copy of this
motif in their promoters (59).
Recently, a novel transcription factor, alternatively
named HAC1 (HAC 5 homologous to ATF and CREB) or
ERN4 (ERN 5 ER nuclear signaling pathway), was identified that binds the UPRE E-box motif during ER stress
(19, 57). The HAC1 gene is required for activation of the
UPR, since yeast strains deleted in this gene no longer
induce the response. Activity of HAC1 is regulated in an
unusual and complex manner, about which there is some
debate in the literature. Two groups have shown that the
HAC1 mRNA is spliced at a nonconsensus splice site
upon induction of the UPR (19, 33). Splicing occurs within
the coding region so that the spliced mRNA gives rise to
a different protein. Unspliced HAC1 mRNA encodes a
230-amino acid protein. Splicing removes the sequence
encoding the last 10 amino acids and adds a novel COOH
terminus of 18 amino acids, giving rise to a 238-amino acid
Hac1p (19, 33). In transient transfection assays, the 238amino acid Hac1p has an ;12-fold higher transcriptional
transactivation capacity than the 230-amino acid form of
the protein (33). Expression of the spliced form of Hac1p,
the 238-amino acid protein, constitutively activates the
UPR. Only this larger 238-amino acid protein can be detected in Western blots; the shorter 230-amino acid form
is not found. Likewise, the Hac1 protein is only detectable
after induction of the unfolded protein response; it is not
present in unstressed yeast cells.
There was considerable disagreement in the literature over the regulation of HAC1 splicing. Cox et al. (19)
proposed that although the 230-amino acid Hac1p is constitutively produced, it is a highly unstable protein that is
rapidly degraded by the ubiquitin-proteasome pathway
and therefore has no effect in the cell. They suggested
that the spliced 238-amino acid Hac1p, in contrast, is a
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FIG. 1. Unfolded protein response (UPR) in
yeast. Presence of mis- or unfolded proteins in
endoplasmic reticulum (ER) is sensed by a yet
unknown mechanism. This leads to dimerization and activation of Ire1p kinase, which autophosphorylates. COOH terminus of Ire1p has
endonuclease activity that becomes activated
and splices HAC1 mRNA. Spliced HAC1 is religated by tRNA ligase RLG1. Upon exit from
nucleus, HAC1 mRNA is translated into Hac1p
transcription factor. Hac1p translocates to nucleus and binds UPR element (UPRE), transactivating expression of UPR target genes, ER resident enzymes which aid in protein folding.
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HEIKE L. PAHL
in tRNA splicing, is used. A mutation in RLG1 [rlg-100, a
C442T point mutation, which changes a conserved histidine (His-148) to tyrosine] completely prevents induction
of the UPR. In these strains, the HAC1 mRNA is not
spliced but rather becomes rapidly degraded. However,
this mutation has no effect on tRNA splicing, proving that
the tRNA ligase encodes two distinct enzymatic activities.
Sidrauski et al. (91) have proposed that tRNA ligase is
required to join the two segments of a cleaved HAC1
mRNA. In the absence of this enzymatic activity, HAC1
becomes cleaved but is then rapidly degraded, since the
second step of splicing, the religation, cannot take place.
Blocking spliceosome-mediated mRNA splicing, in contrast, has no effect on UPR induction (91). Therefore,
HAC1 mRNA is spliced by a novel mechanism requiring
tRNA ligase.
In addition to the transcription factor HAC1, a second component of the UPR signal transduction pathway
has been described. Genetic analyses have identified a
Ser/Thr kinase (Ire1p/Ern1p) whose activity is required
for induction of the UPR in yeast (18, 58). Ire1p acts
upstream of Hac1p, since the HAC1 mRNA is not spliced
in yeast strains lacking the kinase (19). However, the
spliced, 238-amino acid Hac1 is constitutively active, even
in IRE1 mutant strains. Because its structure is similar to
that of mammalian transmembrane receptor kinases, the
following model for Ire1p activation has been proposed
(58, 82): the kinase resides in the membrane of the ER
with its NH2 terminus facing the lumen. This domain acts
as a sensor of ER stress. Mammalian receptor kinases are
activated by dimerization, and Shamu and Walter (83)
have recently shown that Ire1p activity is regulated similarly. Under nonstressed conditions, Ire1p is present as an
inactive monomer. During periods of ER stress, Ire1p
oligomerizes, activating the kinase which trans-autophosphorylates and induces the UPR signaling cascade. In
support of this model, COOH-terminally truncated Ire1p
molecules, which lack the kinase domain, exert a dominant-negative effect (58). These mutants most likely form
kinase-inactive heterodimers with wild-type Ire1p.
Sidrauski and Walter (92) were recently able to show
that Ire1p is a bifunctional enzyme; its COOH terminus
contains, in addition to its kinase activity, an endoribonuclease activity that is responsible for splicing the HAC1
mRNA at both nonconsensus splice sites. The authors
(92) were able to reproduce accurate HAC1 splicing in
vitro, by supplying only Ire1p and tRNA ligase. With this
observation, the entire signal transduction pathway can
now be followed from the ER lumen into the nucleus (Fig.
1). The accumulation of un- or misfolded proteins initiates
Ire1p dimerization, which induces autophosphorylation,
presumably also activating the endoribonuclease activity.
Activated Ire1p, whose COOH terminus can protrude either into the cytoplasm or into the nucleus, cleaves the
HAC1 mRNA. The RLG1 tRNA ligase religates the cleaved
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stable protein and is therefore capable of inducing the
unfolded protein response. In this model, yeast cells constantly translate the unspliced HAC1 mRNA, producing a
very low level of 230-amino acid Hac1p. Upon ER stress,
cells use this small amount of preformed 230-amino acid
Hac1p to initiate the UPR and sustain this response by
producing the more stable 238-amino acid Hac1p through
inducible mRNA splicing (19).
Kawahara et al. (33) were unable to reproduce some
of the results reported by Cox et al. (19). Most importantly, they used point mutants to show that only those
HAC1 mRNA that have undergone splicing are translated
into a protein. Mutation of the 59-splice site, which prevents splicing, completely abrogated protein production
in response to ER stress. Kawahara et al. (33) proposed an
alternative model based on these results. They suggest
that Hac1 expression is controlled posttranscriptionally
through ER stress-induced mRNA splicing, because only
the spliced mRNA is translated into protein.
Recently, a consensus has been reached. It is now
agreed that Hac1p production is regulated at the posttranscriptional level. After reinvestigating their experimental
system, Chapman and Walter (15) now report that both
forms of Hac1, the spliced 238-amino acid form and the
unspliced 230-amino acid form, are equally stable. Furthermore, unspliced Hac1 is not stabilized in yeast strains
deficient in protein degradation, suggesting that absence
of detectable 230-amino acid Hac1 is not due to instability
of the protein. Rather, in their newest study, Chapman
and Walter (15) show that the 252-nucleotide intron in the
unspliced HAC1 mRNA attenuates translation of the protein. Surprisingly, transferring the Hac1 intron to an unrelated green fluorescent protein mRNA also attenuates
translation of this message. Therefore, presence of the
Hac1 intron seems sufficient to prevent translation of a
polyribosome-associated mRNA. Two possible mechanisms have been suggested (15): the intron may prevent
progress of the ribosome either at a distance, by looping
back, or directly, in both cases causing the ribosome to
stall and ultimately release from the mRNA. The newest
model compiled from these data argues that unspliced
HAC1 mRNA is exported from the nucleus, bound to
polyribosomes, but prevented from translation into the
230-amino acid protein by the presence of the intron.
Upon induction of the UPR, newly produced HAC1 mRNA
is spliced and becomes translated into the 238-amino acid
protein, which functions as a transcription factor to induce UPR target genes (15). This model accounts for all
observations made by the two labs investigating Hac1.
Sidrauski et al. (91) have shown that HAC1 splicing
at the nonconsensus splice site occurs via a novel mechanism, distinct from the splicing of other mRNA. Unlike
the previously described mRNA splicing, HAC1 splicing
does not require the spliceosome (91); rather, a tRNA
ligase (RLG1), which has previously only been implicated
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ENDOPLASMIC RETICULUM SIGNAL TRANSDUCTION
ligands can be hypothesized, and all models must now
await experimental verification.
In yeast, inositol-containing phospholipids and lipids
constitute a major proportion of membrane components,
and the level of free inositol regulates phospholipid biosynthesis (111). A fall in the level of free intracellular
inositol induces the transcription of genes encoding enzymes required for phospholipid synthesis. These include
INO1, encoding inositol 1-phosphate synthase, CHO1, encoding phosphatidylserine synthase, and OPI3, encoding
phospholipid methyltransferase. Interestingly, yeast
strains carrying mutations in IRE1, HAC1, RGL1, and
ADA5 are inositol auxotrophs; they require inositol in the
culture medium for growth (18, 91, 110). It is therefore
possible that the UPR coordinately regulates both the
synthesis of ER membranes and the transcription of ERresident proteins. Cox et al. (17) have recently provided
evidence to this effect. They have shown that inositol
starvation induces the transcription of UPR target genes.
The authors demonstrate that this is not because of the
accumulation of underglycosylated proteins under inositol starvation; rather, the UPR is specifically induced by a
decrease in free cellular inositol. The following observations suggest that the transcription of UPR and inositol
target genes may be linked. In response to inositol starvation, IRE1 mutant cells induce much lower levels of
INO1 mRNA than wild-type cells. Although some INO1
mRNA is produced, in IRE1 mutants, cells cannot sustain
mRNA production and therefore never produce the large
amounts required for inositol prototrophy (17). The authors propose a two-step mechanism of INO1 transcriptional activation: an initial Ire1p-independent induction
followed by a second Ire1p-dependent maintenance of
transcription. Interestingly, induction of the UPR by tunicamycin also induces INO1 transcription in an Ire1p- and
Hac1p-dependent manner. Strains lacking either Ire1p or
Hac1p were unable to induce INO1 in response to tunicamycin treatment. Therefore, Ire1p and Hac1p participate in two signal transduction pathways: the UPR and
the inositol response. This redundancy allows cells to
expand both the size and the protein content of their ER
during stress.
2. The UPR in mammalian cells
Induction of the mammalian GRP genes is caused by
a variety of agents including glycosylation inhibitors, reducing agents, heavy metals, amino acid analogs, glucose
starvation, and perturbance of intracellular Ca21 homeostasis (see Table 1) (40, 41, 47). It has been proposed
that the common condition caused by all these treatments
is the accumulation of misfolded proteins in the ER and
that this represents the UPR-inducing stimulus (38). However, recent data by McCormick et al. (52) suggest that
two separate signal transduction pathways may lead to
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ends, producing an open reading frame of 238 amino
acids. This mRNA is translated into 238-amino acid Hac1,
which binds the UPRE, initiating transcription of the UPR
target genes.
Recently, Welihinda et al. (110) identified the first
negative regulator of the UPR. Using a modified yeast
two-hybrid screen, the authors (110) showed that the
serine/threonine phosphatase Ptc2p, a type 2C phosphatase, directly interacts with Ire1p. The interaction is dependent on Ire1p phosphorylation and is mediated by the
kinase interaction domain within Ptc2p. Ptc2p dephosphorylates Ire1p, thereby downregulating the UPR. Consequently, strains carrying null mutations in PTC2 show a
three- to fourfold increase in UPR activity and display
augmented HAC1 splicing. Likewise, overexpression of
wild-type Ptc2p attenuates HAC splicing and downregulates the UPR. Ire1p represents the first identified substrate for type 2C serine/threonine phosphatases.
Welihinda et al. (110) have recently suggested that
Hac1p uses the cofactors Ada2, Ada3, Ada5, and Gcn5,
which form a multisubunit complex, to activate transcription (110). Gcn5 was found to interact with Ire1p in a
modified two-hybrid screen. Subsequently, these data
were corroborated by in vitro coimmunoprecipitation.
Yeast strains deleted in GCN5, ADA2, or ADA3 showed
reduced induction of the KAR2 and PDI1 genes, targets of
the UPR. However, these effects are very weak. Stronger
evidence comes from the observation that strains deficient in ADA5 completely lack KAR2 and PDI1 induction.
In contrast, induction of the heat shock response is normal in these yeast. The Gen5p/Ada2p/Ada3p/Ada5p complex may form a bridge between Hac1p, bound to the
UPRE, around 100 bp upstream of the promoter, and the
TATA-binding protein, bound to the TATA box in the
promoter. However, the significance of these data needs
to be verified.
Although many components of the yeast UPR have
now been identified, one question remains: What is the
sensor of stress in the ER lumen? Somehow the presence
of un- or misfolded proteins must lead to Ire1p dimerization. This step is not well understood, but several models
have been proposed. The GRP78/BiP protein itself has
been suggested as a sensor of ER stress. Because GRP78/
BiP binds to unfolded proteins, thereby preventing their
exit from the ER, the level of free BiP may inversely
correlate with the extent of protein misfolding. In the
simplest model, GRP78/BiP serves directly as a ligand for
monomeric Ire1p, thus preventing oligomerization of the
kinase. As unfolded proteins accumulate, most GRP78/
BiP will form novel complexes with misfolded proteins,
thus allowing Ire1p to oligomerize. In support of this
hypothesis, Morris et al. (61) have shown in mammalian
cells that the overexpression of GRP78/BiP prevents the
induction of GRP transcription. Of course, many other
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ever, the CCAAT element is not sufficient for promoter
activity on its own. In the grp78 promoter, an adjacent
CCAC motif also appears necessary for ER stress induction (73). Therefore, in mammalian cells, no single element seems responsible for ER stress-mediated induction
of transcription; rather, this response is mediated by multiple, functionally redundant elements (112).
The identification of the Ire1p kinase in yeast raises
the question of whether the mammalian UPR is also mediated by a kinase, perhaps even by an Ire1p homolog.
Cao et al. (11) investigated this hypothesis by preparing
stably transfected Chinese hamster ovary cell lines overexpressing yeast Ire1p. Basal and thapsigargin-induced
expression of GRP78 and GRP94 was only increased twofold in these cells. Treatment with okadaic acid, a Ser/Thr
phosphatase inhibitor, increased GRP induction in response to thapsigargin by twofold. In contrast, the tyrosine kinase inhibitor genistein completely inhibited induction of GRP transcription by thapsigargin. The authors
concluded that the mammalian UPR may require both
Ser/Thr and Tyr protein kinases, but these appear not to
be functionally homologous to Ire1p.
Tirasophon et al. (101), however, recently reported
cloning of the mammalian Ire1p homolog. Using a degenerate oligonucleotide from the yeast Ire1p kinase domain,
the authors (101) were able to amplify a PCR fragment
from a human fetal liver cDNA library. With the use of this
fragment as a probe, the entire cDNA was cloned. Like
yeast Ire1p, the human protein contains a transmembrane
domain, a Ser/Thr protein kinase domain, as well as a
domain homologous to the endonuclease RNAse L (101).
Consistent with these observations, human Ire1p autophosphorylates and is able to cleave yeast HAC1 mRNA at
the 59-splice site. Human Ire1p is required for UPR activation as judged by reporter gene assays using the rat BiP
promoter. Overexpression of wild-type Ire1p constitutively activated the reporter gene, whereas expression of
a catalytically inactive form of the protein inhibited ER
stress-induced gene activation in a dominant negative
fashion (101). Cloning of the human Ire1p homolog will
surely prompt the search for a human Hac1.
Recently, Brewer et al. (7) were able to show that
basal transcription of the GRP genes under non-ERstressed conditions is regulated by a novel signal transduction pathway, distinct from the unfolded protein response. This pathway is stimulated by cytokine growth
factors, since cytokine starvation drastically decreased
the basal level of GRP expression. The observation that
the UPR remains intact during starvation and the distinct
kinetics by which the two pathways are activated support
the conclusion that GRP transcription is indeed stimulated by two independent signaling pathways, one for
basic transcription and one, the UPR, for stress-mediated
induction.
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GRP induction in mammalian cells. Many cell types respond to both the glycosylation inhibitor tunicamycin and
the ER Ca21-ATPase inhibitor thapsigargin by increasing
GRP transcription. McCormick et al. (52) reported that
the WEHI72 lymphoma cell lines do not alter GRP transcription after thapsigargin treatment. Interestingly, treatment of these cells with tunicamycin led to a strong
increase in GRP transcription. The authors (52) therefore
propose that two separate signaling pathways, one elicited by ER Ca21 depletion and one by the accumulation of
misfolded or underglycosylated proteins in the organelle,
may induce GRP transcription.
The promoters of mammalian GRP proteins contain a
conserved 28-bp DNA element, the GRP core (14). This
sequence has been shown to bind complex proteins in
electrophoretic mobility shift assays and DNA footprinting analyses. Stress-inducible changes were observed in
the 39-half of the core region by in vivo footprinting
assays, and this sequence was therefore named the stressinducible change region (SICR) (46). A constitutive factor, p70CORE, was first shown to bind this region (46).
p70CORE was recently identified as the transcription factor YY1 (45) and shown to enhance stress induction of the
grp78 promoter. Two additional factors, which bind the
GRP core SICR, were recently identified by southwestern
cloning: YB-1 and dbpA (44). Both are members of the
Y-box family of proteins, which share a conserved DNA
binding motif as well as a cold shock domain. Li et al. (44)
have shown that binding of YB-1 and dbpA to the GRP
core prevents YY1 binding to this site in gel shift assays.
Consistent with this observation, in reporter gene assays,
transfection of the Y-box proteins represses stress induction through the GRP core element. Moreover, YB-1 and
YY1 were shown to interact in yeast-interaction trap assays. However, gel shift assays, transient transfection experiments, and yeast interaction trap all create artificial
conditions for protein-protein or protein-DNA interactions. Therefore, further experiments such as conditional
knockout mice are required to confirm the physiological
role of these proteins in regulating GRP expression.
Despite the fact that the GRP core shares 50% overall
homology to the yeast 22-bp UPRE and contains a stretch
of 80% identity, this sequence does not function as a
UPRE in S. cerevisiae (60). Deletion analysis and linker
scanning of the rat GRP78 promoter showed that the GRP
core element is functionally redundant (112). Its deletion
or mutation does not alter gene induction by misfolded
proteins. Deletion of a CCAAT motif, which binds members of the constitutive CBF/NF-Y family (72), reduces
both basal transcription and stress inducibility of the rat
GRP78 promoter. A similar motif was identified in the
promoter of two additional UPR target genes: GRP94 and
ERp72 (51, 69). Using dominant negative mutants, Zhou
and Lee (117) have shown that the CAAT-binding factor
CBF/NF-Y is required for grp78 promoter activity. How-
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B. A Novel Role for Transcription Factor NFkB
2. ER stress conditions that activate nuclear
factor kB
TABLE
ER overload by protein overexpression
Hepatitis B virus truncated middle HB surface antigen
Influenza virus hemagglutinin
Immunoglobulin m-heavy chain
MHC class I
Adenovirus E3/19K
EPO receptor
Drugs perturbing ER function
Tunicamycin
2-Deoxyglucose
Monensin
Brefeldin A
Thapsigargin
Cyclopiazonic acid
MHC, major histocompatibility.
inhibition of NFkB activation by overexpresssion of a
dominant negative form of IkB renders cells more sensitive to apoptosis by tumor necrosis factor (TNF) as well
as by the chemotherapeutic agent daunorubicin and by
ionizing radiation (106, 107). Thus, in addition to its role
as a mediator of the immune response, NFkB seems to
regulate apoptosis, at least in some cell types.
1. A novel ER-nuclear signaling pathway distinct
from the UPR
All of the previously described activators of NFkB
share two properties: they represent a stress to the cell,
and they act from the outside. We wondered whether an
internally arising stress situation would also activate the
transcription factor. A perturbance in the function of the
ER, the largest cell organelle, could represent such an
internal stress. We observed that expression of the hepatitis B virus protein MHBSt, which accumulates in the ER,
activates NFkB (56). Two models can explain this result:
either NFkB activation is a unique property of MHBSt, or
it represents a more general phenomenon, perhaps related to MHBSt’s accumulation in the ER. We therefore
investigated whether, in general, a perturbance of ER
function would activate NFkB. Indeed, treatment of HeLa
cells with a variety of agents that either inhibit N-glycosylation (tunicamycin and 2-deoxyglucose) or protein
transport by causing the formation of a mixed ER-Golgi
compartment (brefeldin A) strongly induce the transcription factor (64). Moreover, the overexpression of various
membrane proteins, which accumulate in the ER, can
activate NFkB (56, 63, 64, 66) (Table 2). Activation of
NFkB by these agents was observed both in gel mobility
shift assays and by induction of reporter genes controlled
by NFkB DNA binding motifs (56, 63, 64). Supershift
assays revealed that the NFkB complex induced by ER
stress is composed in equal parts of p50/p65 and p50/c-rel
heterodimers (63, 64).
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A number of conditions that perturb ER function
were recently shown to activate NFkB, a transcription
factor previously characterized as a central mediator of
immune and inflammatory responses (64, 66). In a great
variety of cell types, NFkB is found in an inactive, cytoplasmic complex bound to IkB, its inhibitory subunit (1).
Upon exposure of cells to pathological agents, such as
proinflammatory cytokines, antigen, and ultraviolet or
g-irradiation, or upon bacterial and viral infections, NFkB
activity is rapidly induced (for a review, see Baeuerle and
Henkel, Ref. 3). Activation occurs via phosphorylation of
IkB-a on two serine residues, Ser-32 and Ser-36, and
subsequent degradation of the inhibitor by the 26S proteasome (1, 6, 28, 96, 104, 105). Two kinases responsible
for IkB phosphorylation, IKKa and IKKb, were recently
cloned (55, 71, 113, 116). Together with the NFkB-inducing kinase (NIK), they form a high-molecular-weight complex of 700 –900 kDa. The IKKa and IKKb heterodimerize,
but all three kinases appear necessary for IkB phosphorylation, targeting the inhibitor for proteolysis. Degradation of IkB releases the NFkB dimer, which subsequently
translocates to the nucleus, where it activates transcription of its target genes. These include important proinflammatory proteins, such as interferons, cytokines,
chemokines, cell-adhesion molecules, major histocompatibility (MHC) class I molecules and hematopoetic
growth factors (3). A total of five NFkB DNA-binding
subunits, RelA, RelB, c-Rel, p50 and p52, are currently
known. These can hetero- and homodimerize and may
bind distinct NFkB motifs with slightly differing affinities.
This could provide a mechanism for selectively activating
target genes. A total of five IkB proteins control the
activity of NFkB dimers: IkB-a, -b, -g, -d, and -e (3).
Given the nature of its target genes, the major role of
the NFkB system appears to be the induction of a rapid
and coordinated transcription of proinflammatory genes
in response to external, primarily pathogenic stimuli. This
is strongly supported by the phenotype of mice in which
various members of the NFkB family were subject to
targeted disruption (reviewed in Baeuerle and Baltimore,
Ref. 2). For example, mice lacking the c-rel gene fail to
mount effective immune responses to various pathogens,
and their immune cells produce less cytokines in response to stimulation. Likewise, a knockout of the RelA
(p65) subunit causes death early in embryogenesis due to
apoptosis of the liver. In addition, recent experiments
using cell lines derived from mice lacking RelA suggest
that NFkB induction may protect against apoptosis (5).
Fibroblasts and macrophages from these mice undergo
programmed cell death when treated with TNF, whereas
cells from control mice, which can induce NFkB in response to the cytokine, remain viable. These data are
supported by independent experiments that show that
689
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HEIKE L. PAHL
2. Defining the NFkB-activating signal in the ER
The functionally impaired ER must be able to emit
two distinct signals: one that activates NFkB and one that
induces the UPR. A number of stimuli appear to cause the
production of both signals. Because overexpression of a
number of unrelated, wild-type, integral membrane proteins induces NFkB activation (see Table 2), it seems that
the mere accumulation of proteins in the ER membrane
provides an NFkB-activating stimulus. Overexpression of
a bacterial protein in the cytosol (chloramphenicol acetyltransferase) or an insect protein in peroxisomes (firefly
luciferase) had no such effect, arguing against a more
general artifact of transient protein overexpression (64).
We assume that NFkB activation by drugs blocking glycosylation and thus protein exit from the ER, also results
from accumulation of proteins in the ER membrane, overloading the organelle.
The ER-overload hypothesis was investigated using
the adenovirus E3/19K protein as a model (66). Wild-type
E3/19K resides in the ER, where it binds to MHC class I
molecules, thereby preventing their transport to the cell
surface. The viral protein possesses a COOH-terminal
retention signal sequence, which causes the protein to be
continuously retrieved to the ER from post-ER compartments (32). As expected, expression of even small
amounts of E3/19K strongly activates NFkB (66). Because
the sequence requirements of E3/19K for MHC class I
binding and ER retention are precisely known, the NFkBactivating ER signal could be investigated using point
mutants that abolish either property.
Two point mutants that no longer bind MHC class I
molecules activate NFkB as effectively as the wild-type
protein (66). Thus the interaction between E3/19K and
another protein, MHC class I, is not necessary for NFkB
activation. Titration experiments confirmed that the
NFkB-activating effects of overexpressing E3/19K and
MHC class I are additive, not synergistic (66). However,
there is a stringent requirement of ER retention for NFkB
activation. Two mutant proteins, which are equally well
expressed as the wild type, but escape ER retention and
are expressed on the cell surface, no longer activate the
transcription factor even when highly overexpressed (66).
Hence, the sole determinant for the NFkB-activating effects of E3/19K is its ER retention. The dilysine ER retrieval motif in the cytoplasmic tail of E3/19K was not
required, since proteins that carry mutations in this motif
but nonetheless accumulate in the ER were equally potent
in NFkB activation (66). These studies suggest that the
NFkB-activating signal is generated by the accumulation
of proteins within the ER membrane, a process we refer
to as ER overload. At present, we do not know of any
membrane protein that does not activate NFkB when
overexpressed. Therefore, the ER-overload stimulus
seems biophysical in nature rather than dependent on a
particular protein sequence, except, of course, a signal
sequence required for ER import and perhaps a transmembrane domain. We have not yet investigated whether
the overexpression of ER luminal proteins also activates
NFkB. Likewise, it is not clear whether a transmembrane
domain is necessary or may even be sufficient for transcription factor activation. Experiments to answer these
questions are currently being pursued.
C. The ER-Overload Response
1. Messengers of the EOR
Endoplasmic reticulum overload must release a signal from the organelle that reaches NFkB in the cytosol.
Because the ER stores large amounts of Ca21, which can
be released upon stimulation, we investigated whether
this ion serves as a second messenger of the EOR (see
Fig. 2). However, Ca21 had not been previously implicated in NFkB activation, except as a cofactor in T cells.
Nonetheless, two lines of pharmacological evidence suggest that the efflux of Ca21 from the ER is required for
EOR-mediated NFkB activation. First, NFkB induction by
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A number of the stimuli that activate NFkB also
induce the UPR (see Table 1). For example, both NFkB
and the UPR are activated by overexpression of the
m-heavy chain, tunicamycin, 2-deoxyglucose, brefeldin A,
or thapsigargin (64, 66, 82) (Tables 1 and 2). It was therefore important to determine whether NFkB participated
in the UPR or defined a novel, independent signal transduction pathway between the ER and the nucleus.
Several pharmacological and biological inducers distinguish between NFkB induction and UPR activation.
For instance, there are inducers of GRP expression such
as Ca21 ionophores, blockers of cytoplasmic Ca21 increase and 2-mercaptoethanol, that do not activate NFkB
(21, 35, 42, 114). Calcium ionophores and the rise of
cytoplasmic Ca21 have only a costimulatory effect on
NFkB in T cells but cannot activate NFkB in other cell
types, whereas 2-mercaptoethanol even inhibits NFkB activation (81, 102). Likewise, the glucosidase inhibitor castanospermine potently induces GRP expression but not
NFkB activity (64). Conversely, a number of classical
NFkB-inducers, such as TNF-a, do not induce GRP expression and, therefore, act independently of ER stress
(64). Moreover, NFkB is potently induced by the phosphatase inhibitor okadaic acid (OA) (100). Treatment with OA
does not activate GRP transcription on its own but can
potentiate GRP induction in combination with the glycosylation inhibitor thapsigargin (68). Thus a variety of conditions allow a distinction between NFkB and UPR activation, supporting the hypothesis that NFkB functions in
a novel signal transduction pathway between the ER and
the nucleus.
Volume 79
July 1999
ENDOPLASMIC RETICULUM SIGNAL TRANSDUCTION
691
ER stress is prevented by preincubation of cells with two
intracellular Ca21 chelators: 3,4,5-trimethoxybenzoic acid
8-(diethylamino)octyl ester (TMB-8) and BAPTA-AM (65,
66). Second, inhibition of the ER-resident Ca21-ATPase by
thapsigargin or cyclopiazonic acid (CPA), which causes a
rapid efflux of Ca21 from the ER, potently induces NFkB
(66). How the accumulation of proteins in the ER membrane increases its Ca21 permeability is unknown. Perhaps an accumulation of membrane proteins impairs
Ca21-ATPase function. Alternatively, an increased protein-to-lipid ratio may increase the Ca21 permeability of
the lipid bilayer. Both models would explain how a num-
ber of membrane proteins that are completely unrelated
in primary structure can activate the same response.
Calcium is not the sole signal required for NFkB
activation by ER overload. Activation of NFkB by all
inducers tested to date is inhibited by a variety of antioxidants (80, 81). This finding, together with the observation that exogenously applied H2O2 can activate NFkB in
some cell lines (4, 79), suggests that reactive oxygen
intermediates (ROI) can act as second messengers during
NFkB activation. This is also true for NFkB activation by
ER overload. We observed that pretreatment of cells with
a variety of structurally unrelated antioxidants abolishes
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FIG. 2. ER-overload response. Accumulation of wild-type or misfolded proteins in ER
leads to a release of Ca21 from organelle. This
causes production of reactive oxygen intermediates (ROI), which activate transcription factor
nuclear factor kB (NFkB). Activation can be
blocked by antioxidants and by Ca21 chelators,
or it can be activated by drugs that cause an
active release of Ca21 from ER. NFkB induces
transcription of genes involved in inflammatory
and immune responses. CPA, cyclopiazonic
acid.
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HEIKE L. PAHL
TABLE
3. Diseases caused by protein misfolding
Disease
Carbohydrate-deficient
glycoprotein syndrome
a1-Antitrypsin deficiency
Alzheimer’s disease
Cystic fibrosis
Charcot-Marie-Tooth type 1A
Congenital hyperthyroid goiter
Osteogenesis imperfecta
Familial hypercholesterolemia
class 2
von Willebrand disease
Marfan syndrome
Tay-Sachs disease
Retinitis pigmentosa
Leprechaunism
Misfolded Protein
Multiple
a1-Antitrypsin
Presenillin
CFTR
Peripheral myelin protein PMP22
Thyroglobulin
Type I procollagen
LDL receptor
von Willebrand factor
Fibrillin
b-Hexosaminidase
Rhodopsin
Insulin receptor
CFTR, cystic fibrosis transmembrane conductance regulator; LDL,
low-density lipoprotein.
the production of either second messenger reduces the
toxicity of the alkylating agent (48).
2. Physiological Role of the EOR
A) THE ANTIVIRAL RESPONSE. What physiological role does
the EOR serve? Upon viral infection, cells become programmed to produce large amounts of viral capsid proteins
that are processed through the ER. This may elicit an EOR
just as seen upon expression of individual viral membrane
proteins using transient transfection methods. Transient expression of three unrelated viral proteins, influenza hemagglutinin (63), hepatitis B virus MHBSt (56), and adenovirus
E3/19K (66), to levels barely detected by immunofluorescence staining activates NFkB by an EOR (Table 2). Because
the NFkB-activating stimulus appears to simply be the accumulation of membrane proteins, we expect that a large
variety of membrane proteins from unrelated viruses can
elicit this signal. Several NFkB target genes encode important antiviral defense proteins, for example, the cytokine
b-interferon and proteins involved in viral peptide presentation to T cells, such as the proteasome subunit LMP2, the
TAP1 peptide transporter, MHC class I molecules, and b2microglobulin (3). By inducing their expression via NFkB,
the EOR may elicit a fast, nonspecific and therefore broad
antiviral response. Direct evidence for this role of the EOR
remains to be obtained.
B) DISEASES OF PROTEIN FOLDING. Evidence is quickly
growing to suggest that many diseases are caused by
mutations that cause misfolding of proteins (Table 3).
These mutant proteins have been shown to accumulate in
the ER. Interestingly, several of these conditions are accompanied by inflammation, of which the pathomechanism remains unknown. We have hypothesized that the
accumulation of wild-type or mutant proteins in the ER
may lead to NFkB activation. The activated transcription
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NFkB activation by both pharmacological inducers of ER
stress and by viral proteins that accumulate in the organelle (64, 65). At the time we interpreted these data to
mean that ROI also serve as second messengers in ER
overload-mediated NFkB activation. However, the recent
cloning of two IkB kinases has renewed the discussion
about the role of ROI as second messengers in NFkB
activation (55, 71, 113, 116). All signal-transducing proteins required for TNF-mediated NFkB activation have
been cloned (71), and they appear to participate in a
kinase cascade, each activating the next by phosphorylation. Therefore, the role of ROI in this process is not
apparent. Nonetheless, many laboratories have observed
that a variety of structurally unrelated antioxidants inhibit
TNF-stimulated NFkB induction. It is currently not clear
whether ROI indeed serve as second messengers or
whether antioxidants interfere with the activity of one of
the signaling molecules, perhaps the IkB kinase. Alternatively, these agents may act downstream of IkB phosphorylation, perhaps inhibiting ubiquitinylation or degradation
by the proteasome.
Because ER stress-mediated NFkB activation apparently requires two second messengers, Ca21 and ROI, we
wished to determine in what order they are produced and
whether one causes the formation of the other. Two lines of
evidence suggest that the release of Ca21 precedes the formation of ROI during ER overload. First, activation of NFkB
by thapsigargin and CPA, which directly cause a Ca21 release from the ER, is inhibited by the antioxidant pyrrolidine
dithiocarbamate (PDTC) (65). Second, fluorescence-activated cell sorting analysis measuring intracellular peroxide
levels with DCFH showed that treatment of HeLa cells with
either thapsigargin or CPA increases intracellular peroxide
levels. Pretreatment with the Ca21 chelator BAPTA-AM prevented this response, suggesting that the Ca21 release preceded ROI production in these cells (65).
Which enzymes produce ROI in response to Ca21
released from the ER? Various enzymes, such as cyclooxygenases, lipoxygenases, and several oxidases, are
capable of inducibly producing ROI as a side product of
their intrinsic peroxidase activities. Using specific inhibitors for these enzymes, the peroxidase activities of cyclooxygenases and lipoxygenases were identified as possible ROI-producing enzymes during ER stress.
Tepoxalin, a specific inhibitor of peroxidases (98), prevented NFkB activation by thapsigargin but not by TNF
or 12-O-tetradecanoylphorbol 13-acetate (TPA) (65).
Whether tepoxalin inhibits NFkB induction by other ER
stress-eliciting agents remains to be investigated.
Interestingly, another stress-induced signal transduction pathway also involves both the redistribution of intracellular Ca21 and the generation of oxidative stress
(48). Exposure of renal epithelial cells to the alkylating
agent iodoacetamide induces both a rise in intracellular
Ca21 concentration and the generation of ROI. Blocking
Volume 79
July 1999
ENDOPLASMIC RETICULUM SIGNAL TRANSDUCTION
accumulate in the ER of the producing liver cell, rather than
being secreted into the bloodstream. Several different AAT
mutations have been characterized, and their effects vary in
severity. Although some mutations only cause a slight decrease in serum AAT levels, patients homozygous for the PiZ
allele exhibit only 10–15% of normal AAT levels in their
serum and patients carrying “null” mutations, such as Pi Null
Hong Kong, have no detectable serum AAT. The PiZ mutant
has been shown to accumulate in the ER of liver cells,
causing the organelle to enlarge and distend (84, 93). Fifteen
percent of children homozygous for the PiZ mutation develop hepatitis, and one-quarter of these progress to liver
cirrhosis and die before the age of eight (97). To study this
severe disease, transgenic mice carrying the PiZ allele were
generated. These animals suffer neonatal hepatitis and show
accumulation of the mutant AAT protein in the ER of liver
cells (12, 24). We are currently investigating whether the
hepatitis resulting from AAT accumulation is due to ER
overload-mediated NFkB activation and subsequent induction of proinflammatory mediators.
Several additional rare diseases are caused by incorrect folding or processing of proteins in the ER. These
include the carbohydrate-deficient glycoprotein syndrome, which is caused by mutations in carbohydrateprocessing enzymes. As a result, most of the patient’s
proteins are incorrectly glycosylated. The main clinical
manifestation of this disease is a severe malformation of
the nervous system, but these children can suffer from
sterile inflammation of the pericardium, which is often
lethal. Likewise, a group of peripheral neuropathies,
which include the Charcaot-Marie-Tooth disease, are
caused by mutations in the peripheral myelin protein
PMP22. It has been hypothesized that the pathophysiology of these diseases may also involve ER overload-mediated NFkB activation (23).
Medeiros-Neto et al. (54) have described a congenital
hypothyroid goiter with deficient thyroglobulin and have
shown that this disease is due to mutations in the thyroglobulin gene. In four patients, they were able to show
accumulation of thyroglobulin in the ER of thyroid cells,
which was accompanied by a microscopically visible distension of the ER. However, this condition has not been
shown to trigger inflammation of the thyroid gland. Likewise, several other “ER storage diseases,” in which misfolded proteins accumulate in the ER, for example, osteogenesis imperfecta and familial hypocholesterolemia, may
not involve inflammatory processes. Because the discovery of the EOR is rather recent, additional time will be
required to elucidate the role of this novel pathway in the
pathophysiology of ER storage diseases.
D. ER-Nuclear Signaling by SREBP
Cholesterol, an essential cell membrane component,
is made available to cells in two ways. One is the uptake
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factor would lead to expression of its target genes, many
of which encode inflammatory cytokines such as interleukin (IL)-1 and TNF-a and chemokines such as IL-8, monocyte chemattractant protein-1 (MCP-1), monocyte inhibitory protein-1a (MIP-1), and RANTES. Secretion of these
proinflammatory mediators would result in neutrophil,
macrophage, and ultimately T-cell recruitment. These
cells would become activated and in turn themselves
release inflammatory cytokines, stimulating and sustaining the inflammatory process. This model could explain
how the accumulation of a misfolded or a wild-type protein may lead to inflammation; however, it is entirely
hypothetical and needs to be investigated experimentally.
Nonetheless, several examples of diseases caused by
misfolded proteins and accompanied by sterile inflammations are listed (see also Table 3). For example, cystic fibrosis patients develop progressive, cytokine-mediated inflammatory lung disease. Pulmonary inflammation is the major
cause of morbidity and mortality in these patients (36).
Cystic fibrosis is caused by mutations in the cystic fibrosis
transmembrane conductance regulator (CFTR). The most
common mutation, termed DF508, causes the protein to be
entirely retained in the ER (16). A proportion of wild-type
CFTR is also found in the ER; only ;25–50% of the protein
reaches the cell membrane (50). Interestingly, it was recently shown that the airway epithelium, those cells in
which the mutant CFTR accumulates, produce large quantities of inflammatory cytokines (53, 62). We are currently
investigating whether cytokine production is because ER
overload-mediated NFkB activation.
Alzheimer’s disease usually strikes patients in their
70s and 80s. Tragically, there is a familiar form of the
disease that causes an early onset, before the age of 60.
The vast majority of these cases are caused by mutations
in the two recently cloned presenillin genes. Both the
wild-type and the mutant forms of these proteins are
located in the ER (37). Like cystic fibrosis, Alzheimer’s
disease involves the expression of several proinflammatory genes known to be upregulated by NFkB, for example, IL-1, IL-6, and IL-8 (43, 67). Interestingly, PC-12 pheochromocytoma cells expressing the L286V mutant form of
presenillin-1 (PS-1) display increased levels of ROI and
intracellular Ca21 in response to apoptotic stimuli compared with control cells expressing wild-type PS-1 (27). In
addition, thapsigargin treatment of PS-1 mutant cell lines
caused a larger increase in intracellular Ca21 than treatment of cells expressing the wild-type protein. Cells carrying the PS-1 mutant were hypersensitive to the induction of apoptosis by several stimuli. Treatment with
antioxidants or blockers of Ca21 influx relieved mutant
PS-1 cells of this hypersensitivity (27).
a1-Antitrypsin (AAT) deficiency, which affects ;1 in
1,800 live births, is the most common genetic cause of liver
disease in children (99). The deficiency results from mutations that cause the molecule to fold incorrectly and thereby
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HEIKE L. PAHL
Volume 79
of cholesterol in food. After resorption in the small intestine, cholesterol is transported in the bloodstream bound
in low-density lipoproteins (LDL). The LDL receptors on
cell surfaces bind and internalize LDL from the plasma.
Alternatively, almost all cell types can synthesize cholesterol de novo (10). Both cholesterol uptake and synthesis
are subject to feedback repression. Transcription of the
genes encoding the LDL receptor, lipoprotein lipase, as
well as cholesterol and fatty acid biosynthetic enzymes
are inhibited by high intracellular sterol levels and induced upon sterol depletion (26). Regulation is mediated
at the level of transcription. This requires a 10-bp DNA
sequence, the sterol regulatory element 1 (SRE-1), in the
promoters of sterol-regulated genes (94).
A basic-helix-loop-helix-leucine zipper (bHLH-LZ)
protein, SREBP-1, was cloned and shown to activate transcription via the SRE-1 (115). The SRE-1 sequence does
not fit the CANNTG consensus, the so-called E box, described for other bHLH-LZ proteins. Interestingly,
SREBP-1 was cloned independently from an expression
library by virtue of its ability to bind the E-box motif
(103). Spiegelmann and colleagues (34), who named the
protein adipocyte determination- and differentiation-dependent factor 1 (ADD-1), show that the ability of SREBP1/ADD-1 to bind both the SRE and the E box depends on
a unique tyrosine residue at position 335 (34). Mutation of
this amino acid to an arginine, conserved in other bHLHLZ proteins, abolishes binding to the SRE but retains
E-box binding. Conversely, substitution of the consensus
arginine by a tyrosine in another bHLH-LZ protein called
USF confers the ability to bind both sequences on this
heterologous protein (34).
Both SREBP-1 and the homologous SREBP-2 (31) are
synthesized as large precursor molecules of ;1,150 amino
acids, which are inserted into the ER/nuclear membrane
(109) (see Fig. 3). The NH2 terminus is cytoplasmic and
contains the bHLH-LZ motif. The middle of the protein
contains two transmembrane domains, which are inserted
into the ER or nuclear membrane, causing the intervening
30 amino acids to form a “luminal loop” that protrudes
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FIG. 3. Sterol response pathway. Synthesis
and uptake of cholesterol is subject to feedback
repression within cells. How cellular cholesterol
level is sensed is not yet known. Depletion of
cholesterol leads to proteolysis of membranebound transcription factor sterol regulatory element binding protein (SREBP). Proteases that
cleave SREBP at 2 well-characterized sites
within “luminal loop” in ER lumen and within
first transmembrane domain have not been described. SREBP cleavage requires activity of
SREBP cleavage-activating protein (SCAP),
which has been shown to physically interact
with COOH terminus of SREBP. SCAP possesses no enzymatic activity; however, its NH2terminal transmembrane domains may serve as
cellular sterol sensor. Cleaved, active SREBP
transcription factor homodimerizes and activates transcription of genes involved in cholesterol and fatty acid synthesis and uptake. SRE,
sterol regulatory element.
July 1999
ENDOPLASMIC RETICULUM SIGNAL TRANSDUCTION
1. The SREBP cleavage-activating protein
Despite the fact that the proteases remain elusive, a
critical regulator of the SREBP pathway was recently
identified. The SREBP cleavage-activating protein (SCAP)
was cloned from a cell line, which fails to downregulate
SREBP cleavage after sterol saturation. These cells were
shown to have a constitutively activating mutation in
SCAP (29). Like SREBP, SCAP is an integral membrane
protein, since its NH2 terminus contains eight putative
transmembrane domains. Interestingly, these segments
show 55% homology to the transmembrane domains
found in the NH2 terminus of hydroxymethylglutaryl
(HMG)-CoA reductase. In HMG-CoA reductase, the NH2terminal transmembrane region functions as a sterol sensor. The catalytic activity of HMG-CoA reductase is also
regulated by the intracellular sterol concentration. In sterol-depleted cells, the half-life of the enzyme is 10 h. This
decreases dramatically to 1.5 h when cells are saturated
with cholesterol. The NH2-terminal transmembrane domains are responsible for this regulated degradation,
whereas the COOH-terminal cytoplasmic portion bears
the enzymatic activity (39). Although there is no evidence
to suggest that SCAP half-life varies with intracellular
cholesterol levels, it is nonetheless intriguing to suggest
4. Proteins whose expression is activated
by SREBP
TABLE
Proteins involved in cholesterol synthesis
HMG-CoA synthase
HMG-CoA reductase
Farnesyl diphosphate synthase
Squalene synthase
Proteins involved in cholesterol and fatty acid uptake
LDL receptor
Lipoprotein lipase
Proteins involved in fatty acid synthesis
Acetyl-CoA carboxylase
Fatty acid synthase
Stearoyl-CoA desaturase 1
Glycerol-3-phosphate acyltransferase
Membrane components
Caveolin
Regulatory proteins
Leptin
HMG, hydroxymethylglutaryl; SREBP, sterol regulatory element
binding protein.
that its transmembrane domains also serve as a sterol
sensor.
Interestingly, SCAP does not possess any enzymatic
activity, in particular, no protease activity; rather, its
COOH terminus contains four so-called WD domains,
which have been shown to mediate protein-protein interaction in other proteins. Recently, Sakai et al. (75) were
able to show that SCAP and SREBP-2 interact in coimmunoprecipitation experiments and that the COOH terminus
of both proteins is required for complex formation. Interestingly, a COOH-terminal truncation of SREBP-2 leads to
a reduction in proteolytic cleavage, suggesting that a
SCAP/SREBP complex is required for proteolysis and
may therefore represent the target of the SREBP protease.
Cleaved SREBP, which contains the bHLH-LZ motif
and apparently some of the transmembrane domain,
translocates to the nucleus where it activates transcription of target genes. The SRE-1 sites were first reported in
the promoters of the LDL receptor and the HMG-CoA
synthase genes. However, it has recently become clear
that SREBP participates in the transcription of many
more genes. These can be classified into three groups (see
Table 4): 1) genes encoding enzymes involved in cholesterol biosynthesis, such as HMG-CoA synthase; 2) genes
encoding proteins involved in cholesterol and fatty acid
uptake from the plasma, for example, the LDL receptor
and lipoprotein lipase; and 3) genes encoding proteins
mediating fatty acid synthesis, for example, the fatty acid
synthase (FAS) gene. Interestingly, FAS is expressed in a
differentiation-dependent manner in adipocytes (103). Together with the observation that SREBP/ADD-1 mRNA
expression increases substantially during adipocyte differentiation, this raises the intriguing possibility that the
transcription factor exerts a dual function in regulating
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both into the ER and into the nuclear envelope (30, 77).
The COOH terminus again faces the cytoplasm (77). Upon
sterol depletion, SREBP precursors are cleaved by two
sequential proteolytic steps (74), releasing an ;500-amino
acid fragment that contains the basic DNA-binding domain as well as the leucine zipper dimerization motif. The
first cleavage occurs at a Leu-Ser bond in the middle of
the luminal loop (22, 74). Sakai et al. (74) have very
elegantly shown that his step is required before a second
cleavage can take place, this time in the middle of the first
transmembrane spanning region. The first cleavage is sterol dependent: in cells saturated with sterols, proteolysis
of the Leu-Ser bond is abolished. Because the second
cleavage requires the first proteolysis, it also does not
take place in sterol-overloaded cells.
Neither of the proteases that cut SREBP has been
described biochemically, let alone cloned. Both cleavage
sites have been characterized quite carefully, and the
precise amino acid requirements are known (22). The
sequence and location of these sites, one within a luminal
loop and the other within a transmembrane segment,
suggest that these enzymes cleave by unusual mechanisms and may be unlike any proteases known to date.
The cysteine protease called sterol cleavage activator
(SCA) that was shown to cleave SREBP-1/ADD-1 between
the leucine zipper and the first transmembrane domain in
vitro (108) appears not to function as a SREBP cleaving
enzyme in vivo.
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HEIKE L. PAHL
2. Diseases of sterol metabolism
The importance of this regulatory loop becomes apparent in patients with Niemann-Pick disease. Some of
these patients, suffering from the type A and type B
disease, have defects in their acidic sphingomyelinase. In
these patients, shingomyelin is not degraded effectively; it
accumulates and causes a large increase in the amount of
free cholesterol. The patients suffer from cholesterol accumulation, causing as the primary clinical symptom neuronal degradation. The gene whose mutation causes the
distinct Niemann-Pick disease type C was cloned last year
and was shown to have homology to SCAP (13). It shares
the transmembrane domains thought to act as the sterol
sensor. The majority of the mutations identified in Niemann-Pick type C patients affect these transmembrane
domains. These results suggest that NPC1, as the gene
was named, may represent an additional regulator of
cholesterol synthesis. Increased understanding of the regulation of sterol synthesis, for example, the elucidation of
the role of NPC1 and SCAP and the identification of the
proteases that cleave SREBP, should allow the develop-
ment of a specific therapy for these devastating group of
diseases.
3. SREBP in vivo
In addition to the SRE-1, the E box (59-CATGTG-39),
and the ACC promoter site (59-ATCACGTGA-39), SREBP
was recently shown to bind to a fourth sequence, named
SRE-3 (25). This sequence, which is found in the promoter
of the gene for farnesyl diphosphate synthase, shows only
60% identity to either the SRE-1 or the E box. These data
raise the possibility that SREBP may be involved in the
transcription of a plethora of genes mediating cholesterol,
fatty acid, and lipid biosynthesis, which do not contain
consensus SRE-1 binding sites. For example, it has been
pointed out that 13 genes encoding enzymes for lipid
biosynthesis contain a conserved sequence named UASINO, which bears a 60% sequence homology to both
SRE-1 and the E box (25).
All SREBP-regulated promoters studied contain binding sites for additional regulatory factors. Although mutation of the SRE-1 abolishes sterol responsiveness, the
SRE-1 cannot function efficiently on its own (8, 76). In the
LDL receptor promoter, two adjacent binding sites for the
ubiquitous transcription factor Sp1 are required for SRE
function. Both Sp1 and SREBP have been shown to synergistically activate the gene for the LDL receptor (76, 95).
Like the LDL receptor promoter, the ACC promoter also
contains a binding site for Sp1 adjacent to the SREBP
binding site (49). SREBP has recently been shown to
interact directly with the CCAAT-binding factor/nuclear
factor Y (20). The CCAAT boxes are found in both the
HMG-CoA synthase and the farnesal diphosphate synthase promoters.
All results described above were obtained in cultured
cell lines. It therefore remained to be investigated
whether these data could be corroborated in animal models. Perhaps not surprisingly, there are several significant
differences between SREBP function in vitro and in vivo.
For example, in cell lines, proteolysis of SREBP-1 and
SREBP-2 is regulated in parallel; both proteins are
cleaved during sterol deprivation and remain intact when
sterols are saturated. In hamster livers, in contrast,
SREBP-1 is cleaved and active when the animals are fed a
low-fat diet. However, SREBP-2 is not active under these
conditions (85). When these livers are depleted of cholesterol by feeding the animals the cholesterol synthesis
inhibitor lovastatin together with the bile acid-binding
resin Colestipol, SREPB-2 proteolysis is induced (85).
However, contrary to observations in cell lines, SREBP-1
is processed much less effectively under these circumstances. The net result is that livers of hamsters depleted
of cholesterol contain much more active SREBP-2 than
SREBP-1 (85).
Within the two isoforms of SREBP-1, SREBP-1a and
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both sterol responsiveness and adipocyte differentiationspecific gene expression (103).
It has been suggested that because SREBP regulates
both lipid and cholesterol synthesis, both components of
LDL, the use of a single transcription factor may serve to
provide a balanced supply of both components. A similar
hypothesis was made concerning a role for SREBP in
maintaining a balance of fatty acid and cholesterol levels.
It was shown recently that SREBP activates transcription
of the promoter for acetyl-CoA carboxylase (ACC), the
rate-limiting enzyme in fatty acid biosynthesis (49). Because the transcription of HMG-CoA reductase, the ratelimiting enzyme in cholesterol biosynthesis, is also regulated by sterols, the levels of these two cellular lipids may
be regulated coordinately. Perhaps SREBP/ADD-1 also
regulates membrane biosynthesis, since both cholesterol
and fatty acids are essential membrane components.
Recent evidence suggests that SREBP play a critical
role not only in regulating membrane biosynthesis, but in
maintaining its optimal lipid composition. It has been
known since 1980 that sphingomyelin, a membrane lipid,
affects the rate of cholesterol biosynthesis. Addition of
sphingomyelin increases cholesterol synthesis, whereas
its depletion, through treatment with sphingomyelinase,
decreases cholesterol synthesis. Recently, Scheek et al.
(78) were able to show that sphingomyelin depletion in
cultured cells blocks proteolysis of SREBP-2 at the first
site, inside the luminal loop. Thus, in the absence of
sphingomyelin, cholesterol biosynthesis is decreased because SREBP is inactive, preventing transcription of biosynthetic enzymes.
Volume 79
July 1999
ENDOPLASMIC RETICULUM SIGNAL TRANSDUCTION
remains to be investigated whether the low percentage of
surviving animals have compensated for the SREBP-1
deletion by some additional mutation.
III. SUMMARY
The investigation of ER-nuclear signal transduction
has led to the discovery of three distinct signaling pathways: the UPR, the EOR, and the SREBP pathway. Each is
activated by a distinct ER stress signal.
The UPR is induced by the presence of misfolded
proteins in the organelle. In yeast, this stress is sensed by
the Ire1p kinase, which becomes activated and autophosphorylates, thereby inducing its second enzymatic activity, an endonuclease. In unique reactions not previously
described, the Ire1p endonuclease splices the HAC1
mRNA at nonconsensus splice sites. Unlike other characterized mRNA, HAC1 mRNA is religated by the tRNA
ligase Rlg1. Spliced HAC1 mRNA is translated to a 238amino acid protein, which functions as a transcription
factor to induce expression of the UPR target genes.
The EOR is activated by several stimuli that also
induce the UPR, but the common activating signal is the
congestion of the ER with proteins. This causes a release
of Ca21 from the organelle and the production of ROI,
ultimately resulting in the activation of transcription factor NFkB. Because NFkB is a central mediator of the
human immune response, the EOR may play a role in the
pathophysiology of several ER storage diseases.
Cells obtain cholesterol either through uptake or
through de novo synthesis. Both mechanisms are subject
to feedback repression. This is possible because production of both the rate-limiting enzyme of cholesterol biosynthesis and of the LDL receptor, required for cholesterol uptake, is controlled by the same transcription
factor. Activity of this protein, the SREBP, is tightly regulated by cellular sterol concentrations. Inactive SREBP
is inserted into the ER-nuclear membrane as a large precursor molecule. Sterol depletion leads to two sequential
cleavages of the membrane-bound SREBP, releasing an
active transcription factor that activates expression of
genes required not only for cholesterol uptake and biosynthesis but also genes involved in fatty acid synthesis
and uptake. Thus a single transcription factor coordinately regulates the synthesis of two major membrane
components.
Investigation of these three ER-nuclear signal transduction pathways has not only taught us much about how
cells respond to various forms of ER stress. Perhaps more
importantly, they have revealed that these pathways contain regulatory mechanisms unknown in other systems.
The unconventional splicing of the Hac1 transcription
factor may turn out to be the first example of many mRNA
species, not processed by the splicesome. This opens up
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-1c, there are differences between in vitro and in vivo
observations as well. SREBP-1a activates transcription of
the LDL receptor and the HMG-CoA synthase promoters
more potently than does the shorter SREBP-1c, but both
are almost equally active in stimulating the transcription
of the FAS promoter (87). Cells in tissue culture express
much more SREBP-1a than SREBP-1c; some even exclusively produce SREBP-1a (89). However, intact organs,
for example the liver or adipose tissue, express much
higher levels of SREBP-1c than -1a (89). Because of the
different effect of the two isoforms of the transcription
factor on the expression of enzymes responsible for cholesterol synthesis versus those responsible for fatty acid
synthesis, the following hypothesis has been raised. Cell
lines in tissue culture need cholesterol constantly for
membrane synthesis. Therefore, they contain both active
SREBP-1a and SREBP-2, to allow for active cholesterol
synthesis. Cells in vivo, however, only produce a basal
level of active SREBP-1c to support fatty acid synthesis.
Only upon cholesterol depletion do these cells induce
proteolysis of SREBP-2.
The differences in SREBP-1a and SREBP-1c activity
were further demonstrated in transgenic mice, which
overexpress the active form of either protein under the
control of a strong promoter. Whereas animals overexpressing SREBP-1a accumulate large amounts lipids in
their livers, leading to a fourfold enlargement of this
organ (86), animals overexpressing SREBP-1c show only
a 33% increase in liver mass (87). The mRNA for many
SREBP target genes were strongly upregulated in
SREBP-1a transgenic animals, despite the fact that the
accumulation of cholesterol and triglycerides should have
led to a feedback repression (86). In contrast, SREBP-1c
transgenic mice showed no increase in mRNA for cholesterol biosynthetic enzymes (87). The only abnormality in
these animals was a slight, two- to fourfold increase in
mRNA for enzymes responsible for fatty acid synthesis
(87). These enzymes, however, were elevated 20-fold in
mice overexpressing SREBP-1a (86). Therefore, in vivo,
SREBP-1c seems to be responsible for the low level synthesis of mRNA required for fatty acid synthesis, whereas
SREBP-1a is strongly induced following sterol deprivation
to upregulate cholesterol biosynthesis. SREBP-2 transgenic animals are not yet available for analysis.
Recently, mice carrying targeted deletions in the
gene for SREBP-1 were generated (88). Interestingly, animals homozygous for the disruption show two distinct
phenotypes, although they are genetically identical. Although between 50 and 85% of the mice die in utero at day
11 of gestation, the surviving animals are phenotypically
normal. Although they express no SREBP-1, these animals survive and reach adulthood. Their only phenotype
is a slight (1.5-fold) increase in SREBP-2 expression and a
resulting two- to threefold increase in the level of cleaved
SREBP-2 protein found in the nucleus. Consequently, it
697
698
HEIKE L. PAHL
countless possibilities of variation and regulation not previously imagined. Likewise, the unconventional proteolysis of the SREPB transcription may find parallels in other
regulatory pathways. Characterization and cloning of the
unusual proteases involved in SREBP cleavage will indicate whether these proteins uniquely function to regulate
SREBP activity or whether they belong to a family of
proteases that regulates other cellular processes as well.
We look forward to the discovery of additional cellular
regulatory systems, based on the paradigms first discovered while investigating ER-nuclear signaling.
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I express my deep gratitude to Patrick Baeuerle for the
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Thanks also to Alexander Knorre for critical review of the
manuscript.
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