From the
Calreticulin (CRT) is an ubiquitous, highly conserved,
Ca
Calreticulin (CRT)
Based on Ca
Although analysis of the promoter
region of the human CRT gene (11) has revealed several potential
sites that may play an important role in its transcriptional
regulation, none of these have been functionally defined. CRT is
reported to be augmented in response to cellular
proliferation(21, 22) , mitogenic
stimulation(23) , and viral transfection (24) and may be
released from activated neutrophils(25) . Other investigations
have indicated that CRT is a major prompt glycosylation protein in
Chinese hamster ovary cells in response to heat shock(26) . In
this report, we demonstrate, using Northern analyses, mRNA stability
measurements, and nuclear run-on assays, that heat shock also
up-regulates CRT expression in cultured cells predominantly via
transcriptional mechanisms. Furthermore, CAT assays support our
hypothesis that the promoter region of the CRT gene containing a
putative heat shock element (HSE) is sensitive to heat shock stress.
Finally, utilizing rats exposed to heat shock stress, we demonstrate
that both the transcriptional and post-translational glycosylation
responses are active in vivo. These results, and recent
observations indicating the in vivo protective role of heat
shock in sepsis-induced lung injury(27, 28) , suggest
that stress-induced CRT up-regulation may be important in a variety of
biological systems and pathophysiologic conditions.
Fig. 1shows the Northern blot analysis of CRT mRNA in
heat-shocked HUVEC. Following continuous growth of the cells at 37
°C to a postconfluent state, the HUVEC were transferred to an
incubator preset at 42 °C, while control cell monolayers were
maintained at 37 °C. The specific mRNA for CRT had risen
approximately 4.2-fold (see Fig. 3) following 6 h of heat shock
as quantitated by densitometry, and, although some attenuation was
noted, an elevation was maintained for at least 36 h during continuous
heat stress. The pattern of rise of CRT mRNA was similar to that seen
with HSP70 mRNA expression under the same stress conditions (Fig. 1). HSP70 mRNA levels were undetectable under nonstress
conditions, but increased over 100-fold from 1.5 to 6 h following heat
shock. mRNA levels of the ``housekeeping'' gene,
glyceraldehyde-phosphate-3`-dehydrogenase, were unaltered by the
stress, indicating the specific nature of the heat shock response (Fig. 1) and equal loading of total RNA/lane.
Fig. 7(top) is a
Western immunoblot depicting total CRT expression within the lung
tissue as a result of heat shock. In this representative experiment,
lung-derived CRT was notably increased 12 and 18 h following heat
shock, as compared with that from the sham-treated control animal or
animals not exposed to heat shock or anesthesia (not shown).
Furthermore, a second larger band detected by the specific anti-CRT
antibodies was also evident in the immunoblot, consistent with heat
shock-induced glycosylation of CRT observed in vitro by other
investigators(26) . The increase in expression of CRT protein
was paralleled by a persistent increase by 3-fold (by densitometry) in
specific CRT mRNA (Fig. 7) from the same tissue samples. We can
conclude, therefore, that heat shock stress induces transcription and
translation of CRT that is not only manifested in cell culture but that
is also evident in vivo.
In this report, we have shown that heat shock results in
increased expression of CRT predominantly at the level of
transcription. The physiological significance of this finding was
confirmed by in vivo experiments; CRT mRNA and protein are
elevated in the lung tissue of heat-shocked rats. Additionally, the
previously observed early glycosylation response of CRT to hyperthermia (26) was also evident in our in vivo experiments.
In
response to heat shock, CRT transcription transiently increased
3-4-fold followed by a recovery phase in which gene transcription
attenuated and eventually returned toward pre-heat shock levels. The
ubiquitous nature of this CRT response was evident from our observation
that different cells (HUVEC, A549, SKLU-1, MRC-5, and HMEC) responded
to the stress by enhancing transcription of the CRT gene and
accumulation of its mRNA. Our data further suggested that a major
mechanism of CRT mRNA accumulation as depicted in the Northern blots,
was via enhanced transcription, with little or no contribution from
alterations in message stability.
Opas et al.(21) previously reported that in a cultured rat myogenic
cell line, heat shock of 45 °C for 3 h followed by return to 37
°C for 2-48 h did not result in an increase in the levels of
CRT protein. Indeed, a transient reduction in CRT was observed during
the recovery phase between 6 and 8 h. The apparent discordance of our
results with those of Opas et al.(21) might be
explained by the use of different cultured cells or alternatively by
the high temperature used in their experiments, which may have
abrogated the transcriptional response. This possibility is consistent
with observations that the transcriptional response of some
``classic'' heat shock proteins may be suppressed, and indeed
diminished, by particularly large increases in temperature(45) .
The human CRT gene has recently been cloned(11) . Several
putative regulatory sequences were identified using human HeLa cell
nuclear extracts, gel mobility shift analyses, and DNA footprint
localization studies. The possibility that CRT might provide a
housekeeping function similar to the role of other
``chaperones'' was clearly recognized by these
investigators(11) . Our analysis of the region between
-172 and -158 of the human CRT gene revealed the presence
of a putative HSE. Strong biochemical evidence indicates that this
sequence motif, i.e. arrays of two or more inverted repeats of
the five-base pair sequence nGAAn, provides a site of interaction with
heat shock transcription factors. The arrays may function even with
imperfect five-base pair units where there are substitutions for some
of the consensus GAA bases (41, 42, 43) or if
the repeats are variably oriented (44). As noted in Fig. 6, the
identified consensus sequence, gGAAccCAGcgTTCc, within the 5`-flanking
region of the CRT gene, has two perfect inverted repeats with an
intervening imperfect pentad. We propose that this putative HSE in the
CRT gene may be responsible for heat shock-dependent expression of CRT
via transcriptional mechanisms. Our CAT assays support this hypothesis.
However, further CAT studies using genetically altered DNA sequences
within the putative HSE are indicated for confirmation as to the
significance of this region. Furthermore, although the HSE may confer
stress inducibility, the multiple putative cis- and/or trans-regulatory
elements (11) may also confer a range of transcriptional
responses, and consequently the kinetics and magnitude of DNA binding
of heat shock transcription factors to the HSE may be
regulated(46) .
HSPs, often referred to as molecular
chaperones, are currently believed to function as protectors of cells
from harmful effects of a variety of stresses and represent one of the
most conserved families of proteins in evolution (see Welch et al.(47) and Morimoto et al.(48) for reviews).
Following exposure of rats to heat stress for 2-3.5 h, we
observed a persistent increase in lung CRT mRNA and protein, the latter
characterized by the presence of an apparently alternatively
glycosylated form(26) . Recent in vivo studies have
demonstrated the protective property of hyperthermia and heat shock
proteins in sepsis-induced lung injury in rats (27, 28) and endotoxin-mediated mortality in
mice(49) . Our findings of an increase in CRT mRNA and protein
in lung tissue following heat shock, and the persistence of this
response several hours after the removal of the stress, suggests that
CRT also may play a protective role during and following fever or a
variety of other physiological stresses.
Although CRT is ubiquitous
to all cells except erythrocytes, our studies were restricted to
vascular endothelial and lung-derived tissue and cells. We and others
have recently suggested that the stress response may protect the
vasculature by transcriptionally up-regulating important vascular
endothelial cell proteins, including thrombomodulin (40),
thrombospondin(50) , and HSP70(51) . It is therefore
reasonable to hypothesize that CRT may also contribute to this process
in the vasculature. However, it should be noted that considerable
variation in organ expression of HSP72 has been observed in studies by
Hotchkiss et al.(49) when mice were exposed to heat
shock and endotoxin. Consequently, further studies will be required to
evaluate the organ and tissue specificity of the response of CRT to
heat shock stress.
The mechanism(s) by which CRT may play a
physiologically protective role is currently unknown. In addition to
its Ca
A549 cells were transiently co-transfected with pCRT-CAT and pSVTKGH
as described in the methods, and exposed to either 37 °C or 42
°C for the noted times. Results with associated standard deviations
reflect the average of 3 experiments each performed in duplicate,
corrected for transfection efficiency, and relative to activity when
cells were maintained at 37 °C for the same period of time.
-binding protein of the sarcoplasmic and
endoplasmic reticulum. The precise function(s) of CRT is unknown.
However, based on sequence analyses and observations that it may bind
to steroid receptors and integrins and store Ca
within the cell, it has been postulated to play a
``housekeeping'' role. To determine whether the level of
expression of CRT is affected by stress, we examined the heat shock
response of CRT from a variety of cultured cells, including vascular
endothelial, lung epithelial, and lung fibroblasts. Following exposure
of the cells to 42 °C, CRT mRNA transiently accumulated
2.5-4.2-fold at 1-6 h. Nuclear run-on studies and mRNA
stability experiments confirmed that the predominant mechanism of
augmentation was transcriptional. Chloramphenicol acetyltransferase
assays further indicated that the promoter region, containing a
putative heat shock element between -172 and -158 of the
human CRT gene, is heat shock-sensitive. Finally, we demonstrated the in vivo significance of these findings by exposing rats to
hyperthermia. This resulted in accumulation of CRT mRNA and an
augmentation of CRT protein in lung tissue. We hypothesize that this
stress-induced up-regulation of CRT contributes to the mechanism(s) by
which the vascular endothelium and lung tissue, and possibly other
organ systems, maintain homeostasis when exposed to a variety of
pathophysiological conditions.
(
)is a
Ca
-binding protein that was first described in the
sarcoplasmic reticulum of skeletal muscle (1, 2) and
subsequently found to be a major Ca
-binding protein
of the lumen of endoplasmic reticulum (ER) membranes(3) . The
nucleotide sequences of cDNAs encoding CRT from several diverse species
have since been
determined(4, 5, 6, 7, 8, 9, 10, 11, 12) ,
and the deduced amino acid sequences reveal a remarkably high degree of
conservation. CRT is an ubiquitous protein, synthesized by all
mammalian cells tested except
erthrocytes(9, 13, 14) . In spite of
considerable advances in elucidating the structure and tissue
distribution of CRT, less is known about its precise function or
regulation.
-binding properties of the
protein, CRT has been proposed by several laboratories to be a major
Ca
-binding/storage protein of the endoplasmic
reticulum (9). The protein has two distinct
Ca
-binding sites (high affinity/low capacity and low
affinity/high capacity)(13) . Recent investigations have
indicated that CRT might also be important in protein-protein
interactions within the cell. Guan et al. (15) reported a
tight association of flavin-containing lung monooxygenases with CRT,
while Rojiani and co-workers (16) demonstrated interactions
between the integrin
subunit and CRT. A set of endoplasmic
reticulum proteins has also been identified that interact with
CRT(17) . Furthermore, Burns et al.(18) and
Dedhar et al.(19) have provided evidence that CRT may
interact with steroid hormone receptors leading to the modulation of
their transcriptional activity. Finally, CRT has also been implicated
to play a role in the pathogenesis of autoimmune
disorders(10, 20) .
Materials
Goat-anti-rabbit CRT antibodies have
been fully characterized (3, 8) and shown not to
cross-react with calsequestrin (3). Restriction enzymes were from
Boehringer Mannheim Canada (Dorval, Quebec). P-Labeled
nucleotides were purchased from ICN Biomedicals (Canada). RNase T1,
yeast tRNA, and DNase 1 were from Life Technologies, Inc. (Burlington,
Ontario, Canada); NTPs were from Pharmacia Biotech. Inc. (Baie
d'Urfe, Quebec, Canada); and endothelial cell mitogen was from
Biomedical Technologies Inc. (Stoughton, MA).
Cell Culture
HUVEC were harvested by the method of
Jaffe et al.(29) , grown, and characterized as reported
previously (30). Experiments were performed on cells between passages 2
and 5. The immortalized human microvascular endothelial cell line
HMEC-1 were kindly provided by the Biological Products Branch of the
Centers for Disease Control (Atlanta, GA) and cultured as
reported(31, 32) . A549 human lung adenocarcinoma cells,
SKLU-1 human epithelial lung cancer cells, and MRC-5 human diploid
fibroblasts (passage 25-30) were obtained from the American Type
Culture Collection (ATCC) (Rockville, MD) and were grown in
Dulbecco's modified Eagle's medium supplemented with 10%
fetal bovine serum. Media was changed 24 h prior to all experiments.
For the purposes of heat shock, the cell monolayers were first grown in
a humidified atmosphere of 5% CO and 95% air at 37 °C
and were transferred to another humidified, water-jacketed incubator
preset at 42 °C. The incubator was regulated to stay within 0.2
°C of the target temperature. At the appropriate time points, the
cells were immediately placed on ice, washed with ice-cold
phosphate-buffered saline, lysed, and used for membrane preparations,
RNA isolations, or nuclear extractions for run-on assays.
Preparation of cDNA Probes
The full-length
1.9-kilobase cDNA encoding human CRT (33) was a gift of Dr. R.
Sontheimer (South West Medical Center, Dallas, Texas). The plasmid
pGAD28 containing the cDNA for the glycolytic enzyme
glyceraldehyde-3-phosphate dehydrogenase was provided by Dr. Robert J.
Schwartz, Houston, TX(34) , and the cDNA encoding the human heat
shock protein HSP70 was obtained from Dr. Jack Hensold, Case Western
Reserve University, Cleveland, OH. Plasmids were purified by alkaline
lysis and CsCl gradient centrifugation(35) . Following
purification from low melting point agarose gels, the entire
1.9-kilobase cDNA of CRT, the 547-base pair HindIII-XbaI fragment from the coding region of
glyceraldehyde-3-phosphate dehydrogenase, and a 2.3-kilobase BamHI-HindIII fragment encoding HSP70 were used as
probes for Northern blots after labeling with
[-
P]dCTP using the random primer synthesis
method (36). The specific activity of the probes was approximately 2
10
cpm/ng of DNA. For nuclear run-on experiments,
the above cDNA inserts were subcloned into the plasmid Bluescript (pBS)
(Stratagene), and 10 µg of each were dot-blotted onto
nitrocellulose for each condition (see below).
RNA Isolation, Northern analysis, and Measurement of mRNA
Half-life
Total RNA was isolated from confluent cell monolayers
by the method of Chomczynski et al.(37) . Northern
analyses were performed as described previously(38) . To
determine the t of a specific mRNA, postconfluent cells were
exposed to actinomycin D, 25 µg/ml, in culture media for varying
periods of time, after which the cells were lysed for Northern
analysis. Actinomycin D at this concentration totally inhibited
transcription as evaluated by nuclear run-on experiments (data not
shown). Densitometry was performed on scanned autoradiographs
(MacIntosh Color OneScanner) using the NIH Image Analysis Version 1.52
computer program for the MacIntosh.
Isolation of Promoter of Human Calreticulin
Gene
To obtain the CRT promoter region with the putative heat
shock element included, genomic DNA was prepared from approximately 4
10
second passage HUVEC. The following
oligonucleotide primers were synthesized to span the region from
-223 to 22(11) : 1) 5`-gcgtggtcgaccatcat; 2)
5`-cgatctagagcggctctgcagt. Primer 1 is the upstream sense strand, while
primer 2 is the anti-parallel strand between the transcription and
translation start sites and has an XbaI site added to provide
for easier subcloning. To 1 µg of each of the primers, polymerase
chain reaction buffer was added to a final concentration of 10 mM Tris, pH 8.3, 50 mM KCl, 2 mM MgCl
,
0.01% gelatin. After heating to 94 °C, Taq polymerase, 2.5
units (Perkin Elmer Cetus, Mississauga, Canada), was added to initiate
the reaction, and amplification was performed for 30 cycles, with 1-min
primer extensions at 72 °C, denaturation for 30 s at 94 °C, and
primer annealings for 30 s at 45 °C. The resultant 261-base pair
fragment was digested with SalI and XbaI and
subcloned into pBS, and the nucleotide sequence was confirmed by DNA
sequencing. The CRT promoter region was then subcloned into pCAT-Basic
vector (pCAT-BV) (Promega), the latter, which contains the CAT gene, in
the appropriate orientation, to yield pCRT-CAT for CAT assays. pCAT-BV
lacks eukaryotic promoter and enhancer sequences, and expression of
functional CAT activity in transfected cells depends on the insertion
of a functional promoter within the multiple cloning sequence.
Chloramphenicol Acetyltransferase (CAT) and Growth
Hormone Assays
Preconfluent cells in 100-mm dishes were washed
twice with serum-free medium, OPTI-MEM (Life Technologies, Inc.).
Transfection was accomplished by overnight incubation at 37 °C of a
mixture containing 20 µg of the CAT vector, 20 µg of
pSVTKGH(39) , and 25 µg of lipofectin in a total volume of
1.5 ml of OPTI-MEM. pSVTKGH contains the thymidine kinase promoter, an
SV40 enhancer, and the human growth hormone gene. The cells were then
fed with 5 ml of fresh growth medium, 48 h after which heat shock
experiments were started. Following heat shock stress, the cells were
immediately put on ice, and the medium was carefully removed for
quantitation of growth hormone levels by a nonisotopic immunoassay
(system 9670SA from Life Technologies, Inc.). The cell monolayers were
washed 3 times with cold phosphate-buffered saline, scraped, and
pelleted, and lysates were prepared by four freeze-thaw cycles, after
which they were heated at 60 °C for 10 min. CAT reactions using
[H]chloramphenicol were performed overnight at 37
°C in duplicate according to the manufacturer's instructions
(Promega). Transfections with pCAT-BV or without any CAT vector yielded
similar (± 5%) background tritium counts. Experiments were
performed 3 times, and after subtracting the background counts as
determined from parallel transfections with pCAT-BV, the results were
corrected for transfection efficiency by dividing by the respective
growth hormone levels as determined by the immunoassay.
Isolation of Nuclei and Measurement of in Vitro Gene
Transcription
Following heat shock, nuclear pellets from
approximately 5 10
cells/sample were prepared, and
nuclear run-on assays were performed as previously
reported(40) . The
P-labeled RNA specifically bound
to the filters was visualized by autoradiography. Nonspecific
hybridization was determined to be negligible as assessed by using
filters dot-blotted with 10 µg of pBS.
Animal Care and Heat Shock Experiments
All animal
experiments and treatments were approved by the Institutional Animal
Care and Use Committee of the University of Toronto. Sprague-Dawley
rats, 250-300 g (Harlan, Indianapolis, IN), were anesthetized
with pentobarbital 35 mg/kg intraperitoneally and placed into a
prewarmed 41 ± 0.5 °C ambient temperature, humidified
neonatal incubator (Isolette infant incubator, Air-Shields Inc.,
Harboro, PA). The temperature of each animal, which was continuously
monitored by rectal thermometer, persistently increased until it
reached 41 °C (1-1.5 h). Fifteen minutes later, while
maintaining a temperature between 41 and 42.5 °C, the animals were
transferred to room temperature (21 °C), from which point the
post-heat shock duration was determined. Sham-treated rats were also
anesthetized but were not exposed to hyperthermia. The temperatures of
these rats dropped from a mean of 37.5-36.1 ± 0.5 °C 1
h after anesthesia administration and returned to baseline within
4-6 h. At the end of the post-heat shock period, the rats were
sacrificed by sodium pentobarbital overdose. Thoracotomy was performed,
and the lungs were resected and immediately inserted into liquid
nitrogen and stored at -80 °C for further processing. Samples
were subsequently homogenized in phosphate-buffered saline for
SDS-polyacrylamide gel electrophoresis and Western immunoblotting or
lysed in 4 M guanidium isothiocyanate solution (37) for
total RNA preparation and Northern blot analyses.
Miscellaneous
Protein concentration was determined
using the Bio-Rad Protein Assay (Richmond, CA) according to the
manufacturer's instructions. SDS-polyacrylamide gel
electrophoresis and transfer of proteins to nitrocellulose membranes
were carried out as described earlier(38) . CRT was identified
with the goat anti-CRT antibodies by overnight incubation at room
temperature in a buffer containing 20 mM Tris, pH 8.0, 0.15 M NaCl, 5% Carnation milk and detected with alkaline
phosphatase-conjugated rabbit anti-goat antibody. Statistical analyses
were conducted by standard techniques with the aid of StatView computer
program for the MacIntosh (Abacus Concepts Inc., CA). The means are
provided with associated standard deviations (S.D.).
Figure 1:
Northern blot analysis of HUVEC in
response to heat shock. Postconfluent HUVEC were exposed to continuous
42 °C heat shock for 0, 1.5, 6, 24, and 36 h as noted at the top of each lane. 10 µg of total RNA from each
sample were separated on a denaturing formaldehyde-agarose gel as
detailed under ``Experimental Procedures.'' Accumulation of
specific mRNA for glyceraldehyde-3-phosphate dehydrogenase, HSP70, and
CRT was evaluated using the appropriate radiolabeled cDNA probes. The
size of the specific mRNA species is recorded on the left.
Figure 3:
mRNA levels of CRT from different cells
exposed to 42 °C heat shock. Postconfluent cells (HMEC-1, HUVEC,
A549, SKLU-1, MRC-5) were exposed to continuous heat shock for
0-24 h, and 10 µg of total RNA from each sample were
separated on a denaturing formaldehyde-agarose. Northern blot analyses
were performed, detecting with radiolabeled cDNA probes for CRT and
glyceraldehyde-3-phosphate dehydrogenase. Densitometry was performed as
detailed under ``Experimental Procedures,'' and CRT mRNA
levels were quantitated relative to the non-heat shocked levels (100%)
and corrected for glyceraldehyde-3-phosphate dehydrogenase levels at
each time point. The results reflect the mean of three separate
experiments.
We examined the
specificity of the heat shock response by exposing other cultured cells
to hyperthermia. These included human lung epithelial carcinoma A549
cells, human lung adenocarcinoma SKLU-1 cells, an immortalized line of
human microvascular endothelial cells (HMEC-1)(31, 32) ,
and human diploid lung fibroblasts. Although variabilities in the rate
and degree of response were observed, specific CRT mRNA levels in each
of these cell lines as evaluated by Northern blot analyses (Fig. 2) and densitometry (Fig. 3) rose 2.5-3.6-fold
following 1.5-6 h of exposure to hyperthermia and, in all but the
A549 cells, remained elevated at 24 h. These results suggested that the
factors active in up-regulating transcription of the CRT gene are not
specific to a single cell type but rather are probably functional in
most cell biologic systems.
Figure 2:
Northern blot analysis of A549 cells in
response to heat shock. Post-confluent A549 cells were exposed to
continuous 42 °C heat shock for 0, 1.5, 6, 18, and 24 h as labeled
across the top of the figure. mRNA levels of
glyceraldehyde-3-phosphate dehydrogenase (GAPDH), HSP70, and
CRT were evaluated as in Fig. 1.
Given the above results, we determined
whether alterations in CRT mRNA stability were contributing to the heat
shock-induced augmentation in expression. This was done by evaluating
the effect of hyperthermia on actinomycin D-induced, time-dependent
decrease in CRT mRNA. Postconfluent HUVEC were incubated at either 37
or 42 °C in the presence or absence of the RNA polymerase inhibitor
actinomycin D, 25 µg/ml, at which dose the incorporation of
[H]thymidine was suppressed by over 95% within 30
min of exposure, and in vitro transcription was abrogated. In
a representative experiment, densitometry analyses of the Northern
blots shown in Fig. 4indicated that the t of CRT mRNA
when the cells were maintained at 37 °C was 8 h, while the t when the cells were exposed to heat shock was 7.5 h. The lack of a
significant alteration in the stability of the CRT mRNA by heat shock,
suggested that the major mechanism of up-regulation of the message was
via transcription.
Figure 4:
Stability of CRT mRNA as affected by heat
shock. Postconfluent HUVEC were incubated with actinomycin D at 37
°C (A) or 42 °C (B) for 0, 8, 18, or 30 h.
Northern blot analyses were performed as detailed under
``Experimental Procedures'' to determine the stability of the
specific mRNAs for CRT and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
We directly evaluated the in vitro transcriptional response of CRT to heat shock stress by performing
nuclear run-on studies. HSP70 transcripts from HUVEC maintained at 37
°C were almost undetectable. Transcription of the HSP70 gene was
augmented over 100-fold by the 42 °C heat shock (Fig. 5) in a
similar kinetic pattern of expression as its specific mRNA (Fig. 1). CRT gene transcription was also up-regulated 3.5-fold
by the 42 °C heat shock stress within 90 min as quantitated by
densitometry (Fig. 5) and relative to glyceraldehyde-3-phosphate
dehydrogenase gene transcription. This was followed by gradual
attenuation over the ensuing 24 h. These findings were consistent with
the Northern blot analysis (Fig. 1-3) and also supportive
of a predominantly transcriptional response of the CRT gene to the
stress.
Figure 5:
Nuclear run-on studies of CRT and HSP70
genes in HUVEC exposed to heat shock. HUVEC were exposed to continuous
42 °C heat shock for 0, 1.5, 6, and 24 h, and in vitro transcription was determined by nuclear run-on assays as detailed
under ``Experimental Procedures'' and represented by
autoradiography (top), and graphically (bottom), the
latter representing the levels of CRT transcription evaluated by
densitometry of the autoradiograph, corrected for
glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
transcription.
On examination of the promoter region of the human CRT gene
between -172 and -158(11) , we have identified a
putative HSE composed of three inverted repeats of the consensus pentad
nGAAn (Fig. 6). This region may provide a site of interaction
with heat shock transcription factors (41, 42, 43, 44) to mediate the observed
heat shock sensitivity of CRT. The putative HSE within the CRT gene was
therefore examined for response to heat shock by inserting the region
from -223 to 22 in front of a CAT construct (pCRT-CAT). A549
cells were transfected with the pCRT-CAT or pCAT-BV, the latter as a
negative control, and exposed to 37 or 42 °C for varying periods of
time. Growth hormone assays and transfections with pCAT-BV were
performed to correct the CAT activity results for transfection
efficiency and nonspecific background as detailed in the methods. As
seen in , pCRT-CAT expression was augmented by over 3-fold
following 5 h of heat shock stress, indicating that the promoter region
containing the putative HSE is important for heat shock sensitivity of
the CRT gene.
Figure 6:
5` region of the human CRT gene. Underlinedboldface tandem pentamers represent
putative heat shock element, consisting of two perfect repeats flanking
a central imperfect repeat. Putative TATA box is in underlinedboldfaceitalics. Four CCAAT sequences are in boldfaceuppercaseitalics, and
two putative Sp1 binding sites are in boldfacelowercaseitalics. Identification of TATA, CCAAT, and
Sp1 sites, and numbering of human CRT gene is from McCauliffe et
al. (11).
In order to confirm that the CRT gene is up-regulated in vivo, we exposed anesthetized rats normally maintained at
room temperature (21 °C) to heat shock. Immediately following
anesthesia, rats were exposed to an ambient temperature of 41 °C
for 60-90 min until the rectal temperature reached 41-42.5
°C for 15 min. The heat-shocked rats (n = 5) were
then returned to room temperature during which time the rectal
temperature dropped to 38.6 ± 0.3 and 37.8 ± 0.6 °C
at 30 and 60 min post-heat shock, respectively. The rectal temperatures
all returned to base line within 90 min, and no episodes of hypothermia
were documented for 18 h post-heat shock. Consequently, the rats were
hyperthermic for a total duration of approximately 2-3.5 h. 12
and 18 h post-heat shock exposure, the rats were sacrificed for lung
tissue analyses. Control sham-treated animals were not exposed to heat
stress but did develop transient hypothermia to a mean minimum
temperature of 36.1 °C 1 h following the anesthesia. Rectal
temperatures of these animals were also normal by 4-6 h after
administration of anesthesia.
Figure 7:
In vivo expression of CRT protein and mRNA
in rat lung tissue following heat shock. Rats were anesthetized and
warmed in an incubator as detailed under ``Experimental
Procedures.'' The animals were sacrificed at 12 and 18 h after
heat stress administration. The sham-treated control rat (C)
(no heat shock) was sacrificed at 12 h. Lung tissue was homogenized for
SDS-polyacrylamide gel electrophoresis or lysed for RNA preparation.
For CRT protein detection (top), 20 µg of tissue
lysate/lane was electrophoresed by SDS-polyacrylamide gel
electrophoresis and transferred for immunoblotting with a specific
anti-CRT antibody. Specific CRT mRNA levels (bottom) from the
rats' lung tissue was detected with radiolabeled CRT cDNA after
Northern blotting as detailed under ``Experimental
Procedures.''
-binding properties, CRT also interacts with
the DNA-binding domain of the glucocorticoid receptor and other nuclear
hormone receptors(18, 19) , inhibiting
glucocorticoid-sensitive gene expression and retinoic acid-dependent
differentiation. Consequently, stress-induced up-regulation of CRT
could modulate the steroid sensitivity of different cells, thereby
protecting cells/organisms from different stresses. This regulatory
mechanism may therefore have far-reaching effects during a variety of
physiological processes, including differentiation, development, and
homeostasis.
Table: CAT activity of CRT promoter in A549 cells
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.