Medical Research Council Group in Molecular Biology of Membranes, Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2S2
We have isolated and characterized a 12-kb
mouse genomic DNA fragment containing the entire
calreticulin gene and 2.14 kb of the promoter region.
The mouse calreticulin gene consists of nine exons and
eight introns, and it spans 4.2 kb of genomic DNA. A
1.8-kb fragment of the calreticulin promoter was subcloned into a reporter gene plasmid containing
chloramphenicol acetyltransferase. This construct was
then used in transient and stable transfection of NIH/
3T3 cells. Treatment of transfected cells either with the
Ca2+ ionophore A23187, or with the ER Ca2+-ATPase
inhibitor thapsigargin, resulted in a five- to sevenfold increase of the expression of chloramphenicol acetyltransferase protein. Transactivation of the calreticulin
promoter was also increased by fourfold in NIH/3T3
cells treated with bradykinin, a hormone that induces
Ca2+ release from the intracellular Ca2+ stores. Analysis of the promoter deletion constructs revealed that
A23187- and thapsigargin-responsive regions are confined to two regions (115 to
260 and
685 to
1,763) in the calreticulin promoter that contain the
CCAAT nucleotide sequences. Northern blot analysis
of cells treated with A23187, or with thapsigargin, revealed a fivefold increase in calreticulin mRNA levels.
Thapsigargin also induced a fourfold increase in calreticulun protein levels. Importantly, we show by nuclear run-on transcription analysis that calreticulin gene
transcription is increased in NIH/3T3 cells treated with
A23187 and thapsigargin in vivo. This increase in gene
expression required over 4 h of continuous incubation
with the drugs and was also sensitive to treatment with
cycloheximide, suggesting that it is dependent on protein synthesis. Changes in the concentration of extracellular and cytoplasmic Ca2+ did not affect the increased
expression of the calreticulin gene. These studies suggest that stress response to the depletion of intracellular Ca2+ stores induces expression of the calreticulin
gene in vitro and in vivo.
ALTERATIONS in intracellular Ca2+ concentration regulate a variety of diverse cellular functions including
secretion, contraction-relaxation, cell motility, cytoplasmic and mitochondrial metabolism, and protein synthesis and folding (Pozzan et al., 1994 Calreticulin is an unusual luminal ER protein. Several
unique functions have been postulated for the protein, including modulation of gene expression (Burns et al., 1994 In the present study we describe isolation of the mouse
calreticulin gene, including 2.14 kb of its promoter region.
Using reporter genes we show that this promoter is sensitive to the ER Ca2+ store depletion. We demonstrate that,
in mouse fibroblasts, both the Ca2+ ionophore A23187 and
the ER Ca2+-ATPase inhibitor thapsigargin stimulate expression of the calreticulin gene. We also show, by the nuclear run-on transcription assay, that calreticulin gene is
activated by A23187 and thapsigargin in vivo. Over 4 h of
continuous treatment with these drugs was required to induce calreticulin expression, and this stimulation was sensitive to cycloheximide, suggesting that the Ca2+ store depletion-dependent induction of calreticulin expression requires new protein synthesis. Importantly, transactivation of calreticulin promoter was also induced by bradykinin treatment of NIH 3T3 cells. Our results suggest that
stress response to the depletion of intracellular Ca2+ stores
plays a very important role in the regulation of calreticulin gene expression in vitro and in vivo.
Isolation and Sequencing of Genomic Clones
A mouse liver genomic library (a gift from Dr. J. Stone, University of Alberta, Canada) was constructed by partial digestion of genomic DNA
(adult 129/J male) with the restriction enzyme Sau3A, followed by cloning
into the BamHI site of lambda DASH (Stratagene, La Jolla, CA). Screening of the library was carried out as described by Dower et al. (1992) Various fragments of p1.3 were subcloned into pBluescript and their
nucleotide sequences were determined by the double-stranded dideoxynucleotide method. Sequencing was performed in the DNA Sequencing
Laboratory of the Department of Biochemistry, using DNA sequencer
(model 373A; Applied Biosystems, Foster City, CA). T3, T7, or custom-made primers were used for the sequencing reactions. Synthetic oligodeoxynucleotides were made in the DNA Sequencing Laboratory of the Department of Biochemistry, using a DNA/RNA synthesizer (model 392;
Applied Biosystems).
Plasmid Construction
Plasmid pCM7 was constructed by subcloning a 7-kb HindIII restriction
fragment from p1.3 into the HindIII restriction site of pBluescript. This
fragment contained 1.8 kb of the 5
Different restriction fragments of the calreticulin promoter were subcloned into the promoterless reporter plasmids pCATbasic (CAT expression vector) to generate promoter deletion constructs: pCC0 (2,142-bp
SmaI/StuI restriction fragment of the promoter DNA), pCC1 (1,763-bp
HindIII/StuI restriction fragment), pCC2 (685-bp KpnI/StuI restriction
fragment), pCC3 (415-bp AflII/StuI restriction fragment), pCC4 (260-bp
BamHI/StuI restriction fragment), and pCC5 (115-bp PvuI/StuI restriction fragment).
Cell Culture and Drug Treatment
All cell lines were maintained in DME supplemented with 10% calf serum
at 37°C with 5% CO2 in a humidified incubator. Cells were transferred to
10- or 2-cm tissue culture plates 1 d before drug treatment. Stock solutions
of A23187, thapsigargin, BAPTA/AM (Molecular Probes, Inc., Eugene,
OR), and EGTA/AM (Molecular Probes, Inc.) were prepared in 99.5%
dimethyl sulfoxide and were added to the culture medium as specified in
the text. Control cells were incubated with appropriate volume of 99.5%
dimethyl sulfoxide. Cycloheximide and bradykinin were dissolved in water.
Transient and Stable Transfection
Plasmid DNA was purified by column chromatography (QIAGEN Inc.,
Chatsworth, CA). NIH/3T3 cells were grown in 10-cm dishes and transfected using the calcium phosphate method and a BES (N,N-bis (2-hydroxyethyl)-2-aminoethanesulfonic acid) buffer (Ausubel et al., 1989 For stable transfection, NIH/3T3 cells were cotransfected with reporter
plasmid (8 µg), pSV Cell Extraction and Reporter Assays
Cell extracts were prepared by washing cells with PBS followed by incubation for 15 min at room temperature with 100 µl per 2-cm dish of a lysis
buffer containing 100 mM Tris, pH 7.8, 0.5% NP-40, and freshly added 1 mM DTT. Cell extracts were collected and stored at The level of CAT protein in cell extracts was determined using a CAT
ELISA kit with specific anti-CAT antibodies (Boehringer Mannheim Biochemicals, Indianapolis, IN). Luciferase activity was assessed using 10 µl
of cell lysate and 100 µl of luciferase assay reagent (containing 20 mM Tricine, 1.07 mM MgCO3, 2.67 mM MgSO4, 0.1 mM EDTA, 33.3 mM DTT,
270 µM coenzyme A, 530 µM ATP, and 470 µM luciferin). CAT levels
and luciferase activities in cell extracts were always normalized against
Nuclear Run-on Transcription Assay
Nuclei were prepared from the cells treated for 4 h with 10 µM A23187 or
100 nM thapsigargin. The elongating RNA transcripts were labeled in
vitro with [32P]UTP, isolated, and hybridized to membranes (GeneScreen
Plus) containing slot-blotted single-stranded bacteriophage M13 DNA probes specific for mouse calreticulin gene. The probes were designed to
detect either sense or antisense transcription in the gene region of interest. The following DNA probes were used in the assay, all cloned into
M13mp18 and M13mp19 (Rice et al., 1995 Northern and Western Blot Analysis
Northern blot analysis was carried out as described by Burns et al. (1992) Measurements of Intracellular Ca2+ Concentration in
Cycloheximide- and BAPTA-treated Cells
For measurement of the intracellular Ca2+ concentration in cycloheximide-treated (2 h with 100 µM cycloheximide) or control cells, NIH/3T3
cells (2 × 107 per ml) were loaded for 30 min with 2 µM fura-2/AM under
the conditions preventing sequestration of the dye into subcellular organelles (Demaurex et al., 1992 Isolation and Characterization of the Mouse
Calreticulin Gene
To isolate a calreticulin genomic clone, we first screened a
mouse liver genomic DNA library with a cDNA probe
corresponding to the 5 The mouse calreticulin gene has nine exons and eight introns. The different lengths of these coding and noncoding
regions of the gene are summarized in Table I. The exon-
intron boundaries are highly homologous to the reported
mammalian exon-intron consensus sequences (Table I).
However, there is no typical poly(A) signal in the 3 Table I.
Exons amd Introns of the Mouse Calreticulin Gene
). Ca2+ signals also
trigger gene expression, promote cell cycle progression, and activate apoptosis (Schönthal et al., 1991
; Little et al., 1994
; Ghosh and Greenberg, 1995
). The ER is considered
one of the most important and metabolically relevant
sources of cellular Ca2+ (Pozzan et al., 1994
). Ca2+ is released from the ER by InsP3 receptor/ryanodine receptor Ca2+ release channels and it is taken up by the Ca2+-ATPase (Sorrentino and Volpe, 1993
; Pozzan et al., 1994
; Coronado et al., 1994
). The ER contains a characteristic set of
proteins, resident in the lumen, which terminates with the
KDEL ER retrieval signal (Pelham, 1989
) and may be involved in Ca2+ storage. The most extensively studied of
these proteins are Grp78 (BiP), Grp94, ERp72, protein
disulfide isomerase (PDI), and calreticulin (Pozzan et al.,
1994
). These proteins appear also to be involved in many
other aspects of ER function, including protein synthesis and folding (Gething and Sambrook, 1992
). Recently,
Lee's group has shown that the expression of Grp78 and
Grp94 is induced by various physiological stresses including glucose starvation, heat shock, and changes in intracellular Ca2+ concentration (Little et al., 1994
).
;
Dedhar et al., 1994
; Michalak et al., 1996
), a role in cell adhesion (Coppolino et al., 1995
; Opas et al., 1996
), and
maintenance of intracellular Ca2+ homeostasis including
control of store-operated Ca2+ influx (Liu et al., 1994
; Camacho and Lechleiter, 1995
; Bastianutto et al., 1995
; Mery
et al., 1996
). An important recent finding is that it is the
ER form of calreticulin that modulates gene expression
and cell adhesiveness in mouse L fibroblasts (Michalak et
al., 1996
; Opas et al., 1996
). Calreticulin has chaperone activity (Nigam et al., 1994
; Nauseef et al., 1995
; Wada et al.,
1995
; Peterson et al., 1995
; Sapiro et al., 1996
; Otteken and
Moss, 1996
; Van Leeuwen and Kearse, 1996
; Helenius et
al., 1997
) and it is similar to calnexin, an integral ER membrane protein chaperone (Bergeron et al., 1994
; Michalak,
1996
; Krause and Michalak, 1997
). Calreticulin and calnexin are unusual as chaperones because they function
like lectins and bind specifically to partially trimmed, monoglucosylated, N-linked oligosaccharides (Ware et al.,
1995
; Hammond and Helenius, 1995
; Peterson et al., 1995
;
Sapiro et al., 1996
; Helenius et al., 1997
). Calreticulin also
has an antithrombotic activity (Kubawara et al., 1995
) and
it is detected on the cell surface (Gray et al., 1995
; White et
al., 1995
). The protein plays a role in long term "memory"
in Aplysia (Kennedy et al., 1992
), in cytotoxic T cell function/activation (Burns et al., 1992
; Dupuis et al., 1993
), in
neutrophils (Stendhal et al., 1994), in viral RNA replication (Singh et al., 1994
), in sperm cell function (Nakamura et al., 1993
), and in autoimmunity (Sontheimer et al.,
1993
). To rationalize these diverse functions of calreticulin, it is important to identify and understand the mechanisms that regulate its expression. It is apparent that differential expression of calreticulin will have profound
effects on these seemingly diverse cellular functions. The
human calreticulin gene has been isolated, and the nucleotide sequence analysis of its promoter region has revealed several sites that might play a role in regulation of transcription (McCauliffe et al., 1992
).
Materials and Methods
using
GeneScreen Plus hybridization membrane (NEN, DuPont, Mississaga,
Canada). DNA probes were labeled with [32P]CTP (NEN, DuPont) by
random priming. The first screening of the library, with a 711-bp cDNA
fragment corresponding to the 5
-coding region of mouse calreticulin
cDNA (nucleotides 163-874) (Smith and Koch, 1989
), resulted in the isolation of a pseudogene. A second DNA probe was produced by PCR-driven amplification of mouse genomic DNA using the following primers:
T1 (5
-GCGAA TTC AAA GAG CAG TTC TTG GAC GG-3
) corresponding to nucleotides 137-158 of mouse calreticulin cDNA (underlined)
with a 5
EcoRI restriction site, and T2 (5
-CTGGAT CCA CTC GGA
AAC AGC TTC ACG-3
) corresponding to nucleotides 396-416 (underlined) and a 5
BamHI restriction site. The PCR product was inserted between the EcoRI-BamHI restriction sites of pBluescript, and its nucleotide sequence was confirmed (see below). Screening of the mouse
genomic DNA library with this probe resulted in the isolation of one
clone, designated p1.3. This clone was further characterized by Southern
blotting (Ausubel et al., 1989
).
-flanking region and the entire coding
region of the calreticulin gene. The 1.8-kb promoter fragment was further
subcloned into the promoterless chloramphenicol acetyltransferase
(CAT)1 reporter expression vector pCATbasic and pXP-1 (luciferase expression vector [De Wet et al., 1987
] producing plasmids pCC1 and pLC1, respectively). To generate pCC1, a HindIII/StuI fragment of pCM7 (nucleotides
1,723 to +40 of the calreticulin gene; Fig. 1) was cloned into
the blunt-ended XbaI/HindIII sites of pCATbasic. To generate pLC1, an
SmaI/StuI fragment was subcloned into the SmaI restriction site of pXP-1.
Fig. 1.
The nucleotide sequence of the promoter region of the
mouse calreticulin gene. The nucleotides are numbered with the
putative transcriptional initiation site of the mouse calreticulin
gene at +1 (Smith and Koch, 1989). (Horizontal lines) Putative
binding sites for the DNA binding proteins: AP-2, Sp1, AP-1 and
H4TF-1, and SIF. CCAAT sites (boxed). TATA box (underlined). ATG initiation codon (bold). These sequence data are
available from GenBank/EMBL/DDBJ under accession number
U38249.
[View Larger Version of this Image (63K GIF file)]
). For
transient transfection, 10 µg of reporter plasmid and 10 µg of pSV
-gal (internal control) were used per dish. Cells were incubated with the
precipitated DNA for 16-20 h. After an additional 8-h incubation, cells
were treated with the appropriate drugs for the indicated times, and cell
extracts were prepared and assayed for the reporter genes.
gal (8 µg), and pNEO1 (0.5 µg). After a 24-h incubation, cells were selected for resistance to Geneticin (G418) (600 µg/ml).
After 14 d of growth in the presence of G418, ~200 clones were obtained.
These G418-resistant cells were tested for expression of the reporter gene
and
-galactosidase.
80°C until further use.
-galactosidase activity.
-Galactosidase activity was measured by incubating 20 µl of cell lysate in a covered microtiter plate, at 37°C, with 100 µl
of ONPG (o-nitrophenyl-
-D-galactopyranoside) solution (0.8 mg/ml) and
the OD was measured at 420 nm. Data are reported as means ± SD of
four separate experiments performed in triplicate.
): the mouse calreticulin 5
probe was a 700-bp (from +44 to +744) fragment of the murine calreticulin cDNA; the mouse calreticulin 3
probe was a 630-bp (from +750 to
+1,380) fragment of the murine calreticulin cDNA; the mouse Exon 1 c-myc probe was a 436-bp HindIII-BglII fragment extending from +140
to +576 of the murine c-myc gene; the mouse Intron 1 c-myc probe was an
816-bp BglII-SstI fragment from +700 to +1,516 of the murine c-myc
gene; the glyceraldehyde-3 phosphate dehydrogenase (G3PDH) probe
was a 979-bp fragment from +44 to +1,023 of a human G3PDH cDNA;
the
-actin 5
probe was a 583-bp BamHI-BglI fragment from +483 to
+1,083 (Exon 4-6) of a human
-actin cDNA; the histone H2b probe was
a 300-bp BstEII fragment from +110 to +412 of the chicken histone H2b
gene. Radioactivity hybridizing to each probe was quantitated with a Fujix
BAS1000 Phosphorimager (Fuji Photo Film Co., Ltd., Tokyo, Japan) using MacBAS imaging software.
.
The 711-bp, 5
portion of mouse calreticulin cDNA was used as a probe.
The blots were normalized with a human G3PDH cDNA probe (Clontech
Laboratories, San Diego, CA) (Burns et al., 1992
). The relative abundance of each mRNA was determined using a Fujix BAS1000 Phosphorimager. Cellular extracts were prepared for immunoblotting as described
by Mery et al. (1996)
. Cells were directly lysed into Laemmli sample
buffer followed by sonication (Mery et al., 1996
). Proteins were separated
by SDS-PAGE on 10% polyacrylamide gels as described by Laemmli
(1970)
, and then transferred to nitrocellulose membranes (Towbin et al.,
1979
). Immunoblotting was carried out with goat anti-calreticulin as described by Milner et al. (1991)
.
; Mery et al., 1996
). The cells were washed
twice and fluorescence measurements were performed while cells were
continually stirred and maintained at 37°C. Fura-2 fluorescence was monitored at
ex = 340 nm. To determine effects of BAPTA on the intracellular Ca2+ concentration, NIH/3T3 cells were loaded with both 2 µM fura-2/ AM and 20 µM BAPTA/AM as described by Muallem et al. (1990)
. The basal cytosolic Ca2+ concentrations are reported as means ± SD of four
separate experiments performed in triplicate.
Results
-coding region of mouse calreticulin cDNA. Four clones were isolated and nucleotide sequence analysis revealed that each of them represented an
intronless fragment of the calreticulin gene. The nucleotide sequences of these clones were identical to the nucleotide sequence of the 3
region of calreticulin cDNA, and they were missing introns 6, 7, and 8 that were subsequently found in the calreticulin gene (see below). The 5
regions of these clones did not align with any nucleotide
sequences in the EMBL gene database. We concluded that
these clones correspond to a calreticulin pseudogene. Isolation of the calreticulin genomic clone was achieved by
further screening of the same library, with a genomic
probe that did not hybridize to the pseudogene. Screening of >300,000 plaques resulted in the isolation of a single
clone, designated p1.3. This clone has an insert of 12 kb
that contains the entire calreticulin gene and 2.14 kb of the
5
-untranslated region.
-untranslated region. Nucleotide sequencing of the exons revealed
that they are virtually identical to the mouse cDNA. Specifically, only two nucleotides differ compared with the nucleotide sequence of mouse calreticulin cDNA reported
by Mazzarella et al. (1992)
, and only four differ compared
with the sequence reported by Smith and Koch (1989)
. Importantly, these variations in the nucleotide sequence do
not affect the amino acid sequence of the protein.
Fig. 1 shows the nucleotide sequence of the 1,723-bp
promoter region of the mouse calreticulin gene. The sequence was compared with a database of transcriptional
control elements using MacVector v4.5 software. The following putative regulatory elements were found: a TATA
box (nucleotides 30 to
25), several AP-2 sites (nucleotides
74,
258,
300,
305,
518,
553,
1,091,
1,098,
1,251, and
1,477), GC-rich areas including SpI sites
(nucleotides
76,
303, and
312), AP-1 sites (nucleotides
1,034 and
1,378), an SIF PDGF binding site (nucleotide
404), an H4TF-1 site (
183), and four CCAAT
sequences (nucleotides
194,
207,
1,123, and
1,532),
three of which are oriented in the forward direction. AP-2
and H4TF-1 recognition sequences are typically found in
genes that are active during cellular proliferation, and this
is consistent with the finding that calreticulin expression is
increased in stimulated T cells (Burns et al., 1992
).
Comparison of the Mouse and Human Calreticulin Genes
The genomic organization and nucleotide sequence of the
mouse calreticulin gene are very similar to those reported
for the human gene (McCauliffe et al., 1992). The nucleotide sequences of the mouse and the human gene show
>70% identity (calculated by BESTFIT; Genetics Computer Group software, Madison, WI), with the exception
of introns 3 and 6. There are also remarkable similarities in the lengths of the exons and introns of both genes. Fig. 2 shows a DNA dot matrix analysis of the mouse calreticulin
gene compared with the human gene. The coding regions,
especially, show a high degree of identity, and the only significant differences between the two genes are centered
around introns 3 and 6. In the mouse gene these introns
are approximately twice the size of the corresponding introns in the human gene (897 bp vs 421 bp for intron 3, and
169 bp vs 88 bp for intron 6).
Several putative regulatory elements are found in the
promoter region of both genes. However, there are differences between the two promoters in the position and number of these sites. For example, in the 526-bp promoter region, there are only two CCAAT sites found in the mouse
gene, but four in the human gene (Fig. 1). Three Sp-1 sites
are located in the first 500 bp of the mouse promoter compared with only two in the human gene. In this same region, there are five AP-2 sites in the mouse promoter and
only one in the human gene (McCauliffe et al., 1992). No
SIF PDGF binding site is found in the human gene, but
both promoters contain several poly G-rich sequences.
The Calreticulin Promoter Is Activated by Changes in Intracellular Ca2+ Concentration
The availability of the promoter from the mouse calreticulin gene has allowed us to study how it might regulate transcription. To do this, two different reporter gene systems
were used. The 1.8-kb calreticulin promoter region was
cloned into CAT and luciferase expression plasmids, as
described in Materials and Methods, to generate plasmids
pCC1 and pLC1, respectively. These plasmids were then
used for transient and stable transfection of NIH/3T3 cells.
pSV-galactosidase was used as an internal control. Basal levels of CAT protein and luciferase activity were observed in both stably and transiently transfected NIH/3T3
cells, whereas cells transfected with promoterless control
plasmids showed no detectable CAT protein or luciferase
activity (data not shown). Once cells had been transfected,
we investigated whether alteration of intracellular Ca2+
levels, using either A23187 or thapsigargin, affected the
activity of the calreticulin promoter. A23187, a Ca2+ ionophore, equilibrates any Ca2+ gradient across membranes.
Thapsigargin, however, leads to the depletion of the ER
Ca2+ stores by inhibiting the ER Ca2+-ATPase (Thastrup
et al., 1990
; Ghosh et al., 1991
).
Fig. 3 shows that NIH/3T3 cells stably transfected with
pCC1 and pSV-galactosidase produced five- to sevenfold
more CAT protein after treatment for 16 h with 7 µM
A23187 or 100 nM thapsigargin. NIH/3T3 cells were also
stably transfected with pLC1 and pSV
-galactosidase. When these cells were treated with 7 µM A23187 or 100 nM thapsigargin, a threefold increase in luciferase activity
was observed (Fig. 3 A). Similar results were obtained
with mouse fibroblasts that were transiently transfected
with pCC1 or pLC1. The reason for this difference between the CAT and luciferase reporter systems is not
clear, but it may be related to an inhibitory effect of Ca2+
on luciferase activity (unpublished observations).
To identify regions in the calreticulin promoter that may
be responsible for the A23187- and thapsigargin-dependent activation of the calreticulin gene, we have generated
constructs containing several deletions in the calreticulin
promoter. Fig. 3 B shows that the first 115-bp DNA fragment of the calreticulin promoter was not activated by
A23187 or thapsigargin. The region encompassing 115 to
260 was responsible for two- to threefold A23187- and thapsigargin-dependent induction of the calreticulin promoter (Fig. 3 B). Additional activation of the promoter by
A23187 and thapsigargin was observed within the second
region of calreticulin promoter localized between
685
and
1,763 (Fig. 3 B). These two regions of calreticulin
promoter contain the CCAAT nucleotide motif that, at
least in part, may be responsible for the Ca2+ store depletion-dependent activation of the calreticulin gene as
shown for transactivation of the Grp78 (BiP) promoter
(Wooden et al., 1991
; Roy and Lee, 1995
; Roy et al., 1996
).
The Expression of Calreticulin mRNA and Protein Is Induced by A23187 and Thapsigargin in NIH/3T3 Cells
Treatment of nontransfected cells with A23187 and thapsigargin also led to altered expression of the endogenous calreticulin gene. Specifically, we used Northern blot analysis to measure the relative mRNA levels in NIH/3T3 cells treated with these drugs. Fig. 4 A shows that an approximately four- to fivefold increase in the abundance of calreticulin mRNA was observed in cells incubated with these drugs. There was an approximately fourfold increase in calreticulin protein in NIH/3T3 cells incubated with thapsigargin (Fig. 4 B). This suggests that changes in the level of calreticulin mRNA resulted in changes in calreticulin expression.
The Endogenous Calreticulin Gene Is Activated by A23187 and Thapsigargin in NIH/3T3 Cells
To measure transcription rates of the endogenous calreticulin gene and to determine if the accumulation of calreticulin mRNA was the result of increased transcription due
to A23187 and thapsigargin, nuclear run-on transcription
assays were carried out. NIH/3T3 cells were treated for 4 h
with either A23187 or thapsigargin. Nuclei were prepared
from the drug-treated cells and RNA transcripts initiated
in vivo were elongated in vitro in the presence of
[32P]UTP. The radiolabeled run-on transcripts were hybridized to single-stranded DNAs complementary to either specific calreticulin or control mRNAs (sense probes)
or to antisense RNAs from the same regions (antisense
probes). The probes used detected two regions of the
mouse calreticulin gene (5 and 3
regions). Single-stranded probes for the two regions of the
-actin gene, G3PDH
gene, histone H2b gene, and c-myc gene (intron 1 and
exon 1 regions) were included as controls for levels of
transcription. Fig. 5 shows that both A23187 and thapsigargin induced transcription of calreticulin gene and that
the transcription pattern was consistent with A23187- and
thapsigargin-dependent accumulation of calreticulin mRNA
(Fig. 5). The relative abundance of the calreticulin signal
was determined using Phosphorimager analysis of the
blots shown in Fig. 5. Comparison of the levels of calreticulin gene transcription between control and drug-treated
cells relative to that observed for G3PDH revealed an
approximately twofold increase in the calreticulin signal
(Fig. 5). When levels of calreticulin gene transcription
were compared between control and drug-treated cells relative to
-actin, H2b, and c-myc gene transcription, an approximately sixfold increase in calreticulin signal was observed. Control genes (G3PDH, H2b, and c-myc) were
also induced in the presence of A23187 and thapsigargin but to a lesser extent than that observed for calreticulin
gene (Fig. 5). These results indicate that treatment of the
cells with the Ca2+ ionophore A23187, or with thapsigargin, induces the expression of calreticulin at the transcriptional level in vivo.
The Kinetics of Activation of the Calreticulin Promoter
A23187 and thapsigargin modulate intracellular Ca2+ concentration within seconds. To determine the kinetics of the
Ca2+-dependent activation of calreticulin promoter, time-dependent expression of CAT was measured in NCB1
cells (NIH/3T3 cells stably transfected with pCC1 and
pSV-galactosidase). Two experimental protocols were
used for this analysis. First, the cells were incubated for 2, 4, 8, 12, and 16 h with A23187 or thapsigargin followed by
measurement of CAT expression (Fig. 6, open bars). In
the second protocol, the cells were incubated for 2, 4, 8, and 12 h with the drugs followed by incubation in a drug-free medium to a total of 16 h of incubation for each data
point (Fig. 6, hatched bars). Fig. 6 shows that for both protocols the overall kinetics and magnitude of induction of
CAT expression by A23187 and thapsigargin were similar.
Maximal induction of CAT expression in the NCB1 cells
required 16 h of continuous incubation with both drugs
(Fig. 6), or exposure to the drugs for 4-8 h followed by incubation in a drug-free medium to a total of 16 h (Fig. 6).
These results indicate that the Ca2+ store depletion-dependent activation of the calreticulin promoter is very slow
and that it may require de novo protein synthesis.
In previous experiments with untransfected NIH/3T3
cells (Fig. 4), we found that treatment with A23187 or
thapsigargin induced expression of calreticulin mRNA. To
assess whether or not new protein synthesis is required to
mediate this change, NIH/3T3 cells were pretreated with
100 µM cycloheximide before the addition of either 7 µM
A23187 or 100 nM thapsigargin. Total RNA was then isolated and relative levels of calreticulin mRNA were assessed using Northern blot analysis. Fig. 7 shows that
treatment with cycloheximide reduced the A23187- and
thapsigargin-dependent increase in calreticulin mRNA
levels by ~75%, suggesting that new protein synthesis was
involved in the response. To rule out that these effects
may be due to the cycloheximide-dependent changes in
the intracellular Ca2+ concentrations, we measured cytosolic Ca2+ concentration in the cycloheximide-treated
cells. The basal cytosolic-free Ca2+ concentration was not
changed in the cycloheximide-treated cells and was determined to be 100 ± 8 nM (n = 4) and 110 ± 6 nM (n = 4) in
control and cycloheximide-treated cells, respectively.
Depletion of Ca2+ from the Lumen of the ER Activates the Calreticulin Promoter
To test whether extracellular concentrations of Ca2+ affect
the drug-mediated activation of the calreticulin promoter,
we incubated NCB1 cells in a Ca2+-depleted medium supplemented with EGTA. We found that A23187- and
thapsigargin-dependent activation of the promoter was independent of changes in the extracellular Ca2+ concentration (Fig. 8). These results further indicate that depletion of Ca2+ from ER stores is involved in the activation of the
calreticulin promoter.
The Effects of BAPTA/AM and EGTA/AM on Activity of the Calreticulin Promoter
To test whether activation of the calreticulin promoter by
A23187 and thapsigargin is affected by changes in the cytoplasmic Ca2+ concentration, NCB1 cells were treated
with the membrane-permeable Ca2+ chelators BAPTA/
AM (10 µM) or EGTA/AM (10 µM) before treatment with the drugs. Once BAPTA/AM or EGTA/AM enter
the cytosol, they are cleaved to membrane-impermeable
BAPTA or EGTA. They significantly reduce and maintain a low (resting or below) cytoplasmic Ca2+ concentration (Muallem et al., 1990; Preston and Berlin, 1992
). For
example, we showed that the basal cytosolic Ca2+ concentration in the BAPTA-treated cells was reduced to 65 ± 9 nM (n = 4) as compared with the control cells (100 ± 5 nM (n = 4)). NCB1 cells were loaded with BAPTA/AM
or EGTA/AM for 30 min followed by 16-h incubation with
either A23187 or thapsigargin, in normal or Ca2+-depleted
medium. Fig. 8 shows that in BAPTA-treated cells the A23187- and thapsigargin-dependent activation of the calreticulin promoter was reduced by 50 and 30%, respectively. Depletion of extracellular Ca2+ further enhanced
these effects, especially for cells treated with thapsigargin
(Fig. 8). In contrast, loading of NCB1 cells with EGTA/
AM, a cytoplasmic Ca2+ chelator, had no effect on
A23187- or thapsigargin-dependent activation of the calreticulin promoter (Fig. 8).
The Effects of Bradykinin on Activity of the Calreticulin Promoter
To investigate if there is any transactivation of the calreticulin promoter under more physiological conditions of
Ca2+ store depletion, we treated the NCB1 cells with
bradykinin in Ca2+-free DME supplemented with EGTA.
Bradykinin induces Ca2+ depletion of the intracellular
Ca2+ stores in the NIH/3T3 cells (Hashii et al., 1993). Fig. 9
shows that prolonged treatment of the NCB1 cells with
bradykinin in the absence of the extracellular Ca2+ resulted in a fourfold activation of the calreticulin promoter, suggesting that bradykinin-dependent Ca2+ depletion of
Ca2+ stores activates transcription of the calreticulin gene.
In contrast, incubation of the NCB1 cells with bradykinin
for a shorter period of time (4 h), either in the absence or
presence of the extracellular Ca2+ followed by incubation
in bradykinin-free media, did not activate the promoter
(data not shown).
In this study we have isolated the mouse calreticulin gene and determined its genomic organization. Using a reporter gene assay system, we demonstrated that the calreticulin gene is activated by either thapsigargin-, A23187-, or bradykinin-dependent Ca2+ depletion of intracellular Ca2+ stores both in vitro and in vivo. Importantly, run-on experiments documented that depletion of Ca2+ stores also activate endogenous calreticulin gene.2 Finally, we showed that stress response to ER Ca2+ store depletion results in increased calreticulin mRNA and protein levels, and that this increased expression of the calreticulin gene requires de novo protein synthesis.
To initiate this study we first isolated and characterized
a 12-kb genomic DNA fragment containing the entire
mouse calreticulin gene and 2.14 kb of its promoter region.
This allowed us, for the first time, to compare the nucleotide sequence of the two genes encoding calreticulin. The
mouse gene is highly homologous to the human gene (McCauliffe et al., 1992). With the exception of two introns,
which, in the mouse, are twice the size of their human
counterparts, the exon-intron organizations of these genes are basically identical. This high degree of conservation at
the level of gene organization and its nucleotide sequence
is in keeping with earlier observations that the amino acid
sequences of calreticulin from different species are also
highly conserved (Nash et al., 1994
; Michalak, 1996
). For
example, the amino acid sequence identity of mouse and
human calreticulins is >95% (Smith and Koch, 1989
; McCauliffe et al., 1992
).
To investigate the role of the ER Ca2+ stores in activation of calreticulin gene expression, we used two different
agents, the Ca2+ ionophore A23187 and the ER Ca2+-ATPase inhibitor thapsigargin. We found that these drugs are associated with activation of the calreticulin promoter in
vitro and in vivo, as well as with increased expression of
calreticulin mRNA and protein. Activation of the calreticulin promoter by these drugs is independent of changes in
extracellular Ca2+ concentration. Most importantly, we
show that transactivation of calreticulin promoter is increased in cells treated with bradykinin, a hormone that
induces Ca2+ release from Ca2+ stores in NIH/3T3 cells
(Fu et al., 1992; Hashii et al., 1993
). Bradykinin stimulation of cells leads to activation of protein kinase C. However, the bradykinin-dependent activation of calreticulin promoter was not due to the activation of protein kinase C
since phorbol esters had no effect on transactivation of the
calreticulin promoter as measured using the reporter gene
assay system (unpublished observations).
In this study we used a relatively large fragment of the
calreticulin promoter, which allowed us to identify two regions (115 to
260 and
685 to
1,763) in the promoter
containing unique CCAAT nucleotide motifs that may
play a role in the Ca2+ depletion-dependent activation of
the gene. Similar regions have been identified on the
Grp78 promoter and shown to be responsible for the
thapsigargin- and Ca2+ ionophore-mediated transactivation of the gene (Wooden et al., 1991
; Li et al., 1993
; Roy
and Lee, 1995
; Roy et al., 1996
). This motif may therefore
play a specific role in Ca2+-sensitive regulation of calreticulin genes. It is important to note, however, that CCAAT
element alone may not be sufficient for promoter activation since another cellular promoter, the
2(I) collagen
promoter, which contains a similar motif, is only weakly inducible by Ca2+ depletion signal (Roy and Lee, 1995
;
Roy et al., 1996
). It is unlikely, therefore, that there is a
single element responsible for Ca2+ depletion-dependent
activation of gene expression. We are currently investigating, using gel retardation and site-specific mutagenesis techniques, a precise role of the two regions in the calreticulin promoter in Ca2+ store depletion-dependent activation of the calreticulin gene. Increases in expression of the
calreticulin gene required prolonged exposure to Ca2+
ionophore, thapsigargin, or bradykinin (~4 h), and it was
inhibited by cycloheximide, indicating that the calreticulin
gene belongs to a group of "delayed response" genes that
are activated slowly and typically require new protein synthesis for their expression. The mechanism(s) responsible
for the Ca2+ depletion-dependent activation of calreticulin and other genes is not yet known. One possibility is that
the treatment of cells with A23187, thapsigargin, or bradykinin leads to a brief Ca2+ elevation in the cytoplasm,
which may be sufficient to activate long-term effects on
calreticulin gene expression several hours later. However,
this is unlikely since the short-term exposure of cells to
A23187, thapsigargin, or bradykinin either in the presence
or absence of the extracellular Ca2+ has no effect on the
transactivation of the calreticulin gene. Whether or not
changes in the nuclear-free Ca2+ or nuclear Ca2+ binding
proteins (Bachs et al., 1992
) are involved remains to be determined. Thus, we conclude that induction of the calreticulin gene is likely due to a stress response upon ER Ca2+
store depletion.
Recently, Nguyen et al. (1996) and Llewellyn et al.
(1996)
reported activation of the human calreticulin gene
by Ca2+ and/or Ca2+ store depletion. Llewellyn et al.
(1996)
and Nguyen et al. (1996)
used a relatively short
fragment of the human calreticulin promoter (585 and 504 bp, respectively) and therefore observed only threefold induction of expression of the calreticulin gene. This is likely due to transactivation of the first region of the promoter
identified in the present study. An interesting observation
is that calreticulin promoter is also activated by Zn2+
(Nguyen et al., 1996
). We show that BAPTA, a chelator of
Ca2+ and heavy metals (including Zn2+), partially inhibits
activation of the calreticulin promoter elicited by A23187
or thapsigargin treatment. In contrast, EGTA/AM, which
has a higher specificity for Ca2+ than BAPTA, had no effect on A23187- and thapsigargin-dependent activation of
the gene. This indicates that cytoplasmic Ca2+ may not
play a significant role in the A23187- or thapsigargin-dependent activation of the calreticulin gene. It is tempting to speculate, however, that alterations in cytoplasmic
heavy metals (perhaps Zn2+ concentration) may play a
role in transactivation of the calreticulin gene. Baksh et al.
(1995)
discovered that Zn2+ and Ca2+ profoundly affect
interactions between calreticulin and PDI, the two ER-resident proteins. In the presence of Ca2+ or Zn2+, the two
proteins do not associate (Baksh et al., 1995
). Therefore, change in the Zn2+ concentration may not only transactivate calreticulin gene but, if elevated in the lumen of the
ER, it may also modulate levels of "free" calreticulin and/
or PDI in the lumen of the ER (Krause and Michalak,
1997
). Understanding the significance of Zn2+-dependent
transactivation of calreticulin gene awaits further investigation.
What is the significance of Ca2+ store depletion-dependent transactivation of calreticulin gene? Calreticulin is a
multifunctional protein: it modulates steroid-sensitive
gene expression (Burns et al., 1994; Dedhar et al., 1994
),
cellular adhesiveness (Coppolino et al., 1996; Opas et al.,
1996
), and the store-operated Ca2+ influx (Bastianutto et
al., 1995
; Mery et al., 1996
). Furthermore, the protein is a
unique and unusual chaperone as it interacts specifically
with glucosylated proteins (Hammond and Helenius, 1995
;
Helenius et al., 1997
). It remains to be determined why cells upregulate the expression of calreticulin under these
conditions. It is conceivable that overexpression of calreticulin, a Ca2+ binding protein (Bastianutto et al., 1995
; Mery
et al., 1996
), may be required to overcome depletion of the
ER Ca2+ stores. Alternatively, changes in the intraluminal
ER Ca2+ concentration will affect protein translation,
folding, and posttranslational modification, and increased
expression of calreticulin might be necessary to fulfill requirements for chaperone activity. In this study we show
Ca2+ store depletion-dependent regulation of expression
of calreticulin, a new mechanism responsible for the control of expression of the protein. This will likely have an
important role in the regulation of many cellular processes
that are under the control of calreticulin.
We have recently established that the ER form of calreticulin is responsible for the modulation of steroid-sensitive gene expression (Michalak et al., 1996) and for the
regulation of cellular adhesiveness via upregulation of expression of vinculin (Opas et al., 1996
). Thus, we proposed
that calreticulin may function as a "signaling" molecule
from the lumen of the ER (Mery et al., 1996
; Opas et al.,
1996
; Michalak et al., 1996
; Krause and Michalak, 1997
).
This may be similar to the BiP-dependent ER-nuclear signal transduction described in the yeast (Mori et al., 1993
,
1996
; Cox et al., 1993
; Cox and Walter, 1996
; Sidrauski et
al., 1996
); control of cellular cholesterol homeostasis by
SREBP, an ER integral membrane protein (Wang et al.,
1994
); or ER-dependent activation of the NF-
B (Pahl
and Baeuerle, 1995
; Pahl et al., 1996
; for review see Pahl
and Baeuerle, 1997
). It is conceivable, therefore, that depletion of Ca2+ stores not only acts as an ER-nucleus signaling pathway but it may also, via upregulation of expression of calreticulin as documented in this work, affect the
calreticulin-dependent ER-nucleus/cytoplasm signaling.
This may be a new mechanism of Ca2+-dependent modulation of numerous biological and pathophysiological processes.
Received for publication 20 November 1996 and in revised form 11 March 1997.
Please address all correspondence to Marek Michalak, Department of Biochemistry, University of Alberta, 424 Heritage Medical Research Center, Edmonton, Alberta, Canada T6G 2S2. Tel.: (403) 492-2256. Fax: (403) 492-0886. e-mail: Marek.Michalak{at}ualberta.caWe thank J.L. Busaan for superb technical help. We are indebted to J. Stone for the mouse genomic library; to D. McCauliffe for the fragment of a mouse cDNA used for the initial screening of the genomic library; to A.S. Lee for the Grp78 promoter construct; and to R.D. Sontheimer and D. Capra for making the nucleotide sequence of the human calreticulin gene available to us. We also thank R.E. Milner and K.-H. Krause for critical reading of the manuscript.
This work was supported by grants (to M. Michalak and C. Spencer) from the Medical Research Council of Canada, and from the Heart and Stroke Foundation of Alberta and the Zyma Foundation (to M. Michalak). M. Waser was a postdoctoral fellow of the Alberta Heritage Foundation for Medical Research (AHFMR) and the Swiss National Foundation. N. Mesaeli was a postdoctoral fellow of the Heart and Stroke Foundation of Canada. M. Michalak is a Medical Research Council senior scientist and an AHFMR medical scientist.
CAT, chloramphenicol acetyltransferase; G3PDH, glyceraldehyde-3 phosphate dehydrogenase; PDI, protein disulfide isomerase.
1. | Ausubel, F.M., R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl. 1989. Current Protocols in Molecular Biology. Vols. 1 and 2. |
2. | Bachs, O., N. Agell, and E. Carafoli. 1992. Calcium and calmodulin function in the cell nucleus. Biochim. Biophys. Acta. 1113: 259-270 |
3. |
Baksh, S.,
K. Burns,
C. Andrin, and
M. Michalak.
1995.
Interaction of calreticulin with protein disulfide isomerase.
J. Biol. Chem.
270:
31338-31344
|
4. | Bastianutto, C., E. Clementi, F. Codazzi, P. Podini, F. De Giorgi, R. Rizzuto, J. Meldolesi, and T. Pozzan. 1995. Overexpression of calreticulin increases the Ca2+ capacity of rapidly exchanging Ca2+ stores and reveals aspects of their lumenal microenvironment and function. J. Cell Biol. 130: 847-855 [Abstract]. |
5. | Bergeron, J.G.M., M.B. Brenner, D.Y. Thomas, and D. Williams. 1994. Calnexin: a membrane-bound chaperone of the endoplasmic reticulum. TIBS. 19: 124-128 |
6. |
Burns, K.,
C.D. Helgason,
R.C. Bleackley, and
M. Michalak.
1992.
Calreticulin
in T-lymphocytes. Identification of calreticulin in T-lymphocytes and demonstration that activation of T-cells correlates with increased levels of calreticulin mRNA and protein.
J. Biol. Chem.
267:
19039-19042
|
7. | Burns, K., B. Duggan, A.E. Atkinson, K.S. Famulski, M. Nemer, R.C. Bleackley, and M. Michalak. 1994. Modulation of gene expression by calreticulin binding to the glucocorticoid receptor. Nature (Lond.). 367: 476-480 |
8. | Camacho, P., and J.D. Lechleiter. 1995. Calreticulin inhibits repetitive intracellular Ca2+ waves. Cell. 82: 765-771 |
9. |
Coppolino, M.,
C.Y. Leung-Hagesteijn,
S. Dedhar, and
J. Wilkins.
1995.
Inducible
interaction of integrin ![]() ![]() |
10. |
Coronado, R.,
J. Morrissette,
M. Sukhareva, and
D.M. Vaughan.
1994.
Structure and function of ryanodine receptors.
Am. J. Physiol.
266:
C1485-C1504
|
11. | Cox, J.S., and P. Walter. 1996. A novel mechanism for regulating activity of a transcription factor that controls the unfolded protein response. Cell. 87: 391-404 |
12. | Cox, J.S., C.E. Shamu, and P. Walter. 1993. Transcriptional induction of genes encoding endoplasmic reticulum resident proteins requires a transmembrane protein kinase. Cell. 73: 1197-1206 |
13. | De Wet, J.R., K.V. Wood, M. Deluca, D.R. Helinski, and S. Subramani. 1987. Firefly luciferase gene: structure and expression in mammalian cells. Mol. Cell. Biol. 7: 725-737 |
14. | Dedhar, S., P.S. Rennie, M. Shago, C.Y. Leung-Hagesteijn, J. Filmus, R.G. Hawley, N. Bruchovsky, H. Cheng, R.J. Matusik, and V. Giguere. 1994. Inhibition of nuclear hormone receptor activity by calreticulin. Nature (Lond.). 367: 480-483 |
15. |
Demaurex, N.,
D.P. Lew, and
K.-H. Krause.
1992.
Cyclopiazonic acid depletes
intracellular Ca2+ stores and activates an influx pathway for divalent cations
in HL-60 cells.
J. Biol. Chem.
267:
2318-2324
|
16. |
Dower, N.A.,
M.F. Seldin,
S. Pugh, and
J.C. Stone.
1992.
Organization and
chromosomal locations of Rap1![]() |
17. | Dupuis, M., E. Schaerer, K.-H. Krause, and J. Tschopp. 1993. The calcium binding protein calreticulin is a major constituent of lytic granules in cytolytic T lymphocytes. J. Exp. Med. 177: 1-7 [Abstract]. |
18. | Fu, T., Y. Okano, and Y. Nozawa. 1992. Differential pathways (phospholipase C and phospholipase D) of bradykinin-induced biphasic 1,2-diacylglycerol formation in non-transformed and K-ras-transformed NIH/3T3 fibroblasts. Involvement of intracellular Ca2+ oscillations in phosphatidylcholine breakdown. Biochem. J. 283: 347-354 |
19. | Gething, M.-J., and J. Sambrook. 1992. Protein folding in the cell. Nature (Lond.). 355: 33-45 |
20. | Ghosh, A., and M.E. Greenberg. 1995. Calcium signaling in neurons: molecular mechanisms and cellular consequences. Science (Wash. DC). 268: 239-247 |
21. |
Ghosh, T.K.,
J.H. Bian,
A.D. Short,
S.L. Rybak, and
D.L. Gill.
1991.
Persistent
intracellular calcium pool depletion by thapsigargin and its influence on cell
growth.
J. Biol. Chem.
266:
24690-24697
|
22. |
Gray, A.J.,
P.W. Park,
T.J. Broekelmann,
G.J. Laurent,
J.T. Reeves,
K.R. Stenmark, and
R.P. Mecham.
1995.
The mitogenic effects of the B![]() |
23. | Hammond, C., and A. Helenius. 1995. Quality control in the secretory pathways. Curr. Opin. Cell Biol. 7: 523-529 |
24. |
Hashii, M.,
Y. Nozawa,
Higashida, and
H. .
1993.
Bradykinin-induced cytosolic
Ca2+ oscillations and inositol tetrakisphosphate-induced Ca2+ influx in voltage-clamped ras-transformed NIH/3T3 fibroblasts.
J. Biol. Chem.
268:
19403-19410
|
25. | Helenius, A., E.S. Trombetta, D.N. Hebert, and J.F. Simons. 1997. Calnexin, calreticulin and the folding of glycoproteins. Trends Cell Biol. 7: 193-200 . |
26. | Kennedy, T.E., D. Kuhl, A. Barzilai, J.D. Sweatt, and E.R. Kandel. 1992. Long-term sensitization training in Aplysia leads to an increase in calreticulin, a major presynaptic calcium-binding protein. Neuron. 9: 1013-1024 |
27. | Krause, K.-H., and M. Michalak. 1997. Calreticulin. Cell. 88: 439-443 |
28. |
Kubawara, K.,
D.J. Pinsky,
A.M. Schmidt,
C. Benedict,
J. Brett,
S. Ogawa,
M.J. Broekman,
A.J. Marcus,
R. Sciacca,
M. Michalak, et al
.
1995.
Calreticulin, an
antithrombotic agent which binds to vitamin K+-dependent coagulation factors, stimulates endothelial nitric oxide production, and limits thrombosis in
canine coronary arteries.
J. Biol. Chem.
270:
8179-8187
|
29. | Laemmli, U.K.. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond.). 227: 680-685 |
30. |
Li, W.W.,
S. Alexandre,
X. Cao, and
A.S. Lee.
1993.
Transactivation of the
grp78 promoter by Ca2+ depletion. A comparative analysis with A23187 and
the endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin.
J. Biol.
Chem.
268:
12003-12009
|
31. | Little, E., M. Ramakrishnan, B. Roy, G. Gazit, and A.S. Lee. 1994. The glucose-regulated proteins (GRP78 and GRP94): functions, gene regulation, and applications. Crit. Rev. Eukaryotic Gene Expr. 4: 1-18 |
32. |
Liu, N.,
R.E. Fine,
E. Simons, and
R.J. Johnson.
1994.
Decreasing calreticulin
expression lowers the Ca2+ response to bradykinin and increases sensitivity
to ionomomycin in NG-108-15 cells.
J. Biol. Chem.
269:
28635-28639
|
33. | Llewellyn, D.H., J.M. Kendall, F.N. Sheikh, and A.K. Campbell. 1996. Induction of calreticulin expression in HeLa cells by depletion of the endoplasmic reticulum Ca2+ store and inhibition of N-linked glycosylation. Biochem. J. 318: 555-560 |
34. | Mazzarella, R.A., P. Gold, M. Cunningham, and M. Green. 1992. Determination of the sequence of an expressible cDNA clone encoding ERp60/calregulin by the use of a novel nest set method. Gene (Amst.). 120: 217-225 |
35. |
McCauliffe, D.P.,
Y.-S. Yang,
J. Wilson,
R.D. Sontheimer, and
J.D. Capra.
1992.
The 5![]() |
36. |
Mery, L.,
N. Mesaeli,
M. Michalak,
M. Opas,
D.P. Lew, and
K.-H. Krause.
1996.
Overexpression of calreticulin increases intracellular Ca2+-storage and decreases store-dependent Ca2+ influx.
J. Biol. Chem.
271:
9332-9339
|
37. | Michalak, M. 1996. Calreticulin. R.E. Landes Co., Austin, TX. 207 pp. |
38. |
Michalak, M.,
K. Burns,
C. Andrin,
N. Mesaeli,
G. Jass,
J.L. Busaan, and
M. Opas.
1996.
Endoplasmic reticulum form of calreticulin modulates glucocorticoid-sensitive gene expression.
J. Biol. Chem.
271:
29436-29445
|
39. |
Milner, R.E.,
S. Baksh,
C. Shemanko,
M.R. Carpenter,
L. Smillie,
J.E. Vance,
M. Opas, and
M. Michalak.
1991.
Calreticulin, and not calsequestrin, is the
major calcium binding protein of smooth muscle sarcoplasmic reticulum and
liver endoplasmic reticulum.
J. Biol. Chem.
266:
7155-7165
|
40. | Mori, K., W. Ma, M.J. Gething, and J. Sambrook. 1993. A transmembrane protein with a cdc2/CDC28-related kinase activity is required for signaling from the ER to the nucleus. Cell. 74: 743-756 |
41. |
Mori, K.,
T. Kawahara,
H. Yoshida,
H. Yanagi, and
T. Yura.
1996.
Signaling
from endoplasmic reticulum to nucleus: transcription factor with a basic-leucine zipper motif is required for the unfolded protein-response pathway.
Genes Cell.
1:
803-817
.
|
42. |
Muallem, S.,
M. Khademazad, and
G. Sachs.
1990.
The route of Ca2+ entry during reloading of the intracellular Ca2+ pool in pancreatic acini.
J. Biol. Chem.
265:
2011-2016
|
43. | Nakamura, M., M. Moriya, T. Baba, Y. Michikawa, T. Yamanobe, K. Arai, S. Okinaga, and T. Kobayashi. 1993. An endoplasmic reticulum protein, calreticulin, is transported into the acrosome of rat sperm. Exp. Cell Res. 205: 101-110 |
44. | Nash, P.D., M. Opas, and M. Michalak. 1994. Calreticulin, not just another calcium-binding protein. Mol. Cell. Biochem. 135: 71-78 |
45. |
Nauseef, W.M.,
S.J. McCormick, and
R.A. Clark.
1995.
Calreticulin functions
as a molecular chaperone in the biosynthesis of myeloperoxidase.
J. Biol.
Chem.
270:
4741-4747
|
46. | Nguyen, T.Q., J.D. Capra, and R.D. Sontheimer. 1996. Calreticulin is transcriptionally upregulated by heat shock, calcium and heavy metals. Mol. Immunol. 33: 379-386 |
47. |
Nigam, S.K.,
A.L. Goldberg,
S. Ho,
M.F. Rhode,
K.T. Bush, and
M.Y. Sherman.
1994.
A set of endoplasmic reticulum proteins possessing properties of
molecular chaperones includes Ca2+-binding proteins and members of the
thioredoxin superfamily.
J. Biol. Chem.
269:
1744-1749
|
48. | Opas, M., M. Szewczenko-Pawlikowski, G.K. Jass, N. Mesaeli, and M. Michalak. 1996. Calreticulin modulates cell adhesivness via regulation of vinculin expression. J. Cell Biol. 135: 1913-1923 [Abstract]. |
49. |
Otteken, A., and
B. Moss.
1996.
Calreticulin interacts with newly synthesized
human immunodeficiency virus type 1 envelope glycoprotein, suggesting a
chaperone function similar to that of calnexin.
J. Biol. Chem.
271:
97-103
|
50. |
Pahl, H.L., and
P.A. Baeuerle.
1995.
A novel signal transduction pathway from
the endoplasmic reticulum to the nucleus is mediated by transcription factor
NF-![]() |
51. | Pahl, H.L., and P.A. Baeuerle. 1997. Endoplasmic reticulum-induced signal transduction and gene expression. Trends Cell Biol. 7: 50-55 . |
52. |
Pahl, H.L.,
M. Sester,
H.G. Burgert, and
P.A. Baeuerle.
1996.
Activation of
transcription factor NF-![]() |
53. | Pelham, H.R.B.. 1989. Control of protein exit from endoplasmic reticulum. Annu. Rev. Cell Biol. 5: 1-23 . |
54. | Peterson, J.R., A. Ora, P.N. Van, and A. Helenius. 1995. Calreticulin is a lectin-like molecular chaperone for glycoproteins in the endoplasmic reticulum. Mol. Biol. Cell. 6: 1173-1184 [Abstract]. |
55. |
Pozzan, T.,
R. Rizzuto,
P. Volpe, and
J. Meldolesi.
1994.
Molecular and cellular
physiology of intracellular calcium stores.
Physiol. Rev.
74:
595-636
|
56. | Preston, S.F., and R.D. Berlin. 1992. An intracellular calcium store regulates protein synthesis in HeLa cells, but it is not the hormone-sensitive store. Cell Calcium. 13: 303-312 |
57. | Rice, S.A., M.C. Long, V. Lam, P.A. Schaffer, and C.A. Spencer. 1995. Herpes simplex virus immediate-early protein ICP22 is required for viral modification of host RNA polymerase II and establishment of the normal viral transcription program. J. Virol. 69: 5550-5559 [Abstract]. |
58. | Roy, B., and A.S. Lee. 1995. Transduction of calcium stress through interactions of the human transcription factor CBF with the proximal CCAAT regulatory element of the grp78/BiP promoter. Mol. Cell. Biol. 15: 2263-2274 [Abstract]. |
59. |
Roy, B.,
W.W. Li, and
A.S. Lee.
1996.
Calcium-sensitive transcriptional activation of the proximal CCAAT regulatory element of the grp78/BiP promoter
by the human nuclear factor CBF/NF-Y.
J. Biol. Chem.
271:
28995-29002
|
60. |
Sapiro, R.G.,
Q. Zhu,
V. Bhoyroo, and
H.-D. Söling.
1996.
Definition of the lectin-like properties of the molecular chaperone, calreticulin, and demonstration of its copurification with endomannosidase from rat liver Golgi.
J. Biol.
Chem.
271:
11588-11594
|
61. | Schönthal, A., J. Sugarman, J.H. Brown, M.R. Hanley, and J.R. Feramisco. 1991. Regulation of c-fos and c-jun protooncogene expression by the Ca2+-ATPase inhibitor thapsigargin. Proc. Natl. Acad. Sci. USA. 88: 7069-7100 . |
62. | Senapathy, P., M.B. Shapiro, and N.L. Harris. 1990. Splice junctions, branch point sites, and exons: sequence statistics, identification, and applications to genome project. Methods Enzymol. 183: 252-278 |
63. | Sidrauski, C., J.S. Cox, and P. Walter. 1996. tRNA ligase is required for regulated mRNA splicing in the unfolded protein responses. Cell. 87: 405-413 |
64. |
Singh, N.K.,
C.D. Atreya, and
H.L. Nakhasi.
1994.
Identification of calreticulin
as a rubella virus RNA binding protein.
Proc. Natl. Acad. Sci. USA.
91:
12770-12774
|
65. | Smith, M.J., and G.L.E. Koch. 1989. Multiple zones in the sequence of calreticulin (CRP55, calregulin, HACBP), a major calcium binding ER/SR protein. EMBO (Eur. Mol. Biol. Organ.) J. 8: 3581-3586 [Abstract]. |
66. | Sontheimer, R.D., T.-S. Lieu, and J.D. Capra. 1993. Calreticulin: the diverse functional repertoire of a new human autoantigen. Immunologist. 1: 155-160 . |
67. | Sorrentino, V., and P. Volpe. 1993. Ryanodine receptors: how many, where and why? Trends Pharmacol. Sci. 14: 98-103 |
68. | Stendahl, O., K.-H. Krause, J. Kirschner, P. Jerström, J.-M. Theler, R.A. Clark, J.-L. Carpentier, and D.P. Lew. 1994. Redistribution of intracellular Ca2+ stores during phagocytosis in human neutrophils. Science (Wash. DC). 265: 1439-1441 |
69. | Thastrup, O., P.J. Cullen, B.K. Drobak, M.R. Hanley, and A.P. Dawson. 1990. Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2+-ATPase. Proc. Natl. Acad. Sci. USA. 87: 2466-2470 [Abstract]. |
70. | Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA. 76: 4350-4354 [Abstract]. |
71. |
Van Leeuwen, J.E.M., and
K.P. Kearse.
1996.
The related molecular chaperones calnexin and calreticulin differentially associate with nascent T cell antigen receptor proteins within the endoplasmic reticulum.
J. Biol. Chem.
271:
25345-25349
|
72. |
Wada, I.,
S. Imai,
M. Kai,
F. Sakane, and
H. Kanoh.
1995.
Chaperone function
of calreticulin when expressed in the endoplasmic reticulum as the membrane-anchored and soluble forms.
J. Biol. Chem.
270:
20298-20304
|
73. | Wang, X., R. Sato, M.S. Brown, X. Hau, and J.L. Goldstein. 1994. SREBP-1, a membrane-bound transcription factor released by sterol-regulated proteolysis. Cell. 77: 53-62 |
74. |
Ware, F.E.,
A. Vassilakos,
P.A. Peterson,
M.R. Jackson,
M.A. Lehrman, and
D.B. Williams.
1995.
The molecular chaperone calnexin binds Glc1Man9-GlcNAc2 oligosaccharides as an initial step in recognizing unfolded glycoproteins.
J. Biol. Chem.
270:
4697-4704
|
75. |
White, T.K.,
Q. Zhu, and
M.L. Tanzer.
1995.
Cell surface calreticulin is a putative mannoside lectin which triggers mouse melanoma cell spreading.
J. Biol.
Chem.
270:
15926-15929
|
76. | Wooden, S.K., L.J. Li, D. Navarro, I. Qadri, L. Pereira, and A.S. Lee. 1991. Transactivation of the grp78 promoter by malfolded proteins, glycosylation block, and calcium ionophore is mediated through a proximal region containing a CCAAT motif which interacts with CTF/NF-I. Mol. Cell. Biol. 11: 5612-5623 |