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Effects of extracellular calcium concentration on protein synthesis in Aedes albopictus cells.

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48
Kawamura and Carvalho
Archives of Insect Biochemistry and Physiology 46:48–55 (2001)
This article originally published in Volume 39
Archives of Insect Biochemistry and Physiology 39:47–54 (1998)
Effects of Extracellular Calcium Concentration on Protein
Synthesis in Aedes albopictus Cells
Marcia Tie Kawamura and Maria da Gloria da Costa Carvalho*
Laboratório de Controle da Expressão Gênica, Instituto de Biofísica Carlos Chagas Filho,
Universidade Federal do Rio de Janeiro, Centro de Ciências da Saúde, Ilha do Fundão,
Rio de Janeiro, R.J., Brazil
The influence of extracellular calcium concentration on mosquito cells was investigated in Aedes albopictus cells cultured
in a medium with different amounts of calcium. Protein synthesis in cells incubated in low calcium culture medium was
inhibited when compared to control cells. This inhibition was
reversed by addition of calcium to the culture medium. Two
calcium-induced proteins of approximately 70,000 and 80,000
daltons were detected when calcium was added to the extracellular medium of cells incubated in low calcium medium
for longer than 2 h. Northern-blot analysis indicated that
Hsp70 (heat shock protein of 70,000 dalton) specific mRNA
is present in cells that were cultured in low calcium medium
suggesting that the 70,000 dalton protein is a member of the
Hsp70 family. Our results indicate that extracellular calcium
concentration can modify the gene expression pattern in A.
albopictus cells and the absence of calcium in the culture
medium could be considered a stress factor. Arch. Insect
Biochem. Physiol. 39:47–54, 1998. © 1998 Wiley-Liss, Inc.
Key words: calcium; protein synthesis; Aedes albopictus cells
Contract grant sponsor: Conselho Nacional de Desenvolvitmento Científico e Tecnológico (CNPq); Contract grant
sponsor: Financiadora de Estudos e Projetos (FINEP).
Abbreviations used: ATP = adenosine 5′-triphosphate;
cDNA = complementary deoxyribonucleic acid; D-MEM =
Dulbecco’s modified Eagle medium; EGTA = ethylene glycol bis(β-aminoethyl ether)-N,N,n′,N′-tetraacetic acid; eIF2α = α subunit of eukaryotic protein synthesis initiation
factor 2; Fura-2 = acetoxymethyl ester; Grp78 = glucose
regulated protein of 78 daltons; Grp94 = glucose regulated
protein of 94 daltons; GRPs = glucose regulated proteins; Hepes
= 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HSPs =
heat shock proteins; Hsp70 = 70,000 daltons heat shock pro-
© 2001 Wiley-Liss, Inc.
tein; MEM = Eagle’s minimal essential medium; PAGE = polyacrylamide gel electrophoresis; PBS = phosphate-buffered
saline; PKR = double-stranded RNA-dependent protein kinase; SDS = sodium dodecyl sulfate.
*Correspondence to: Dr. Maria da Gloria da Costa Carvalho,
Laboratório de Controle da Expressão Gênica, Instituto de
Biofísica Carlos Chagas Filho, Universidade Federal do Rio
de Janeiro, Centro de Ciências da Saúde, Ilha do Fundão,
Rio de Janeiro, R.J. 21949-900 Brazil. E-mail: mgccosta@
ibccf.biof.ufrj.br
Received 2 December 1997; accepted 10 August 1998
Calcium in Aedes albopictus Cells
INTRODUCTION
The maintenance of a low cytosolic free-calcium concentration is a common feature of all eukaryotic cells (Pietrobon et al., 1990). Calcium
concentration in the cytoplasm of eukaryotic cells
is approximately 10–7 M, that is 10,000-fold lower
than extracellular concentration (Clampham,
1995). The precise regulation of calcium concentration has been associated with diverse cellular
processes such as secretion, motility, muscular
contraction, cell division, membrane permeability, and changes in gene expression (Carafoli,
1987). Calcium ion (Ca2+) also acts as a second
messenger, and in activating and regulating a variety of enzymes, receptors, ionic channels, and
structural proteins (Berridge, 1991).
Protein synthesis in eukaryotic cells is a complex process that is affected by external influences
(Brostrom and Brostrom, 1990). There is evidence
supporting that Ca2+ may be a prominent factor in
the regulation of protein synthesis in a variety of
eukaryotic cell types (Perkins et al., 1997; Paschen
et al., 1996). Effects of this cation on protein synthesis have been detected in both intact cells and
tissues exposed to low calcium concentration (Perkin
et al., 1997; Brostrom et al., 1984). Brostrom et al.
(1983) observed that the depletion of intracellular
Ca2+ stores induces a concomitant 4–10-fold reduction in the rate of amino acid incorporation in C-6
glial tumor cells. The first evidence for the regulatory role of Ca2+ in the expression of specific genes
was presented by White and Bancroft (1983) who
reported that the addition of Ca2+ to rat pituitary
tumor GH3 cells incubated in Ca2+-free and serumfree medium induced elevated levels of prolactin
mRNA. Activation of c-fos proto-oncogene expression by calcium was described in proliferating cells
and growth factor-stimulated quiescent rat adrenal
pheochromocytoma PC12 cells (Kelly et al., 1983;
Gilchrist et al., 1994). It was subsequently demonstrated that the induction of c-fos expression by
nerve growth factor in the presence of benzodiazepines blocked the calcium channel, suggesting a
Ca2+-dependence of growth factor stimulation
(Curran and Morgan, 1985).
Ca2+ effects on protein synthesis have been
well described in diverse vertebrate cells. Nevertheless, the role of this cation on protein synthesis remains uninvestigated in mosquito cells. To
49
further understand this mechanism, we investigated the effects of extracellular calcium on protein synthesis in Aedes albopictus mosquito cells.
MATERIALS AND METHODS
Cell Cultures
A. albopictus mosquito cells (clone C6/36)
used in this study were a gift from Dr. R.E. Shope,
Arbovirus Research Unit, Yale University, New
Haven, CT (Igarashi, 1978). The cells were cultured in Dulbecco’s modified Eagle medium (DMEM; Gibco, Grand Island, NY) supplemented
with 0.2 mM nonessential amino acids (L-alanine,
L-asparagine, L-aspartic acid, L-glutamic acid, Lglycine, proline, and serine), 2.25% NaHCO3, 2%
fetal calf serum, 8% calf serum, penicillin (500
U/ml), and streptomycin (100 µg/ml). The cells
were grown in glass bottles (35.5 cm2) or in scintillation vials (5 cm2) at 28°C and in an atmosphere of 5% CO2. A confluent culture of cells in
a scintillation vial contained 2 × 106 cells/vial. For
subculture, confluent monolayers (4.0 × 107 cells/
bottle) were gently washed with Dulbecco’s phosphate-buffered saline (PBS), pH 7.2, and after a
short exposure to trypsin, the cells were suspended in the culture medium.
Cell Treatment and Labeling of Cultures With
[35S]-Methionine
A. albopictus cells grown in scintillation vials were incubated in Ca2+-free Eagle’s minimal
essential medium (MEM; Gibco) in the presence
of 0.5 mM EGTA, a calcium chelator (low calcium
medium) or without EGTA and with 0.5 mM calcium (normal calcium medium) for different periods. In the last 20 min of incubation, the MEM
medium was supplemented with 15 µCi/ml [35S]methionine (400 Ci/mmol; ICN Pharmaceuticals,
Costa Mesa, CA). Then, the medium was removed
and the cells in the monolayer were homogenized
and resuspended in 80 µl of electrophoresis loading buffer (6.25 mM Tris-HCl, pH 6.8; 2% sodium
dodecylsulfate [SDS]; 10% glycerol; 5% 2-mercaptoethanol, and 0.001% bromophenol blue).
Analysis of [35S]-Methionine-Labeled Proteins
by Polyacrylamide Gel Electrophoresis
Samples of [35S]-methionine labeled cells in
the loading buffer were heated for 10 min at
50
Kawamura and Carvalho
100°C and the proteins were separated by electrophoresis on one-dimensional 12.5% polyacrylamide
gels using the SDS buffer system of Laemmli
(1970) at room temperature. The approximate
molecular weight of proteins was determined by
comparing their migration rate with those of
coelectrophoresed standard proteins (Pharmacia,
Piscataway, NJ): phosphorylase b, 94,000; bovine
serum albumin, 67,000; ovalbumin, 43,000; carbonic anhydrase, 30,000; soybean trypsin inhibitor, 20,100; α-lactalbumin, 14,400. The dried gels
were exposed to Kodak X-Omat (YAR-S; Kodak,
Rochester, NY) film for 48 h. In all groups of experiments the same amount of protein was loaded
on each gel lane.
Quantification of Protein Synthesis
For quantification of protein synthesis, densitometric tracings of the autoradiograms were
made with an LKB 2202 Ultroscan Laser Densitometer (Pharmacia). The relative protein synthesis quantification was determined by calculating
the areas of densitometric tracings.
RNA Extraction and Northern
Hybridization Analysis
Total RNA was isolated according to Holmes
and Bonner (1973). Briefly, the A. albopictus
cells were lysed with Holmes-Bonner buffer (10
mM Tris-HCl, pH 8.0; 350 mM NaCl; 7 M urea
and 2 g% SDS) and the total RNA were extracted from the lysate with phenol:chloroform
(Sambrook et al., 1989). For Northern analysis, 10 µg of RNA of each sample were denatured in formaldehyde and fractionated on 1.2%
agarose-formaldehyde gel and transferred to nitrocellulose filters (Gibco BRL, Gaithersburg,
MD) by capillary blotting, as described by Thomas (1980). The nucleic acids bound to nitrocellulose filters were baked for 2 h at 80°C in a
vacuum oven and prehybridized in a medium
consisting of 50% formamide, 100 µg/ml denatured salmon sperm DNA, 5 × SSC, 5 × Denhardt’s solution, and 50 mM Na2HPO3, pH 6.8.
Blot hybridization was performed using a nicktranslated [α-32P]dATP labeled pBR322 plasmid
containing the Drosophila melanogaster HSP70
cDNA cloned into Bam HI and Sal I sites (Livak
et al., 1978). Hybridizations were performed at
37°C for 48 h. After hybridization, the mem-
branes were washed 3 times in 2 × SSC and
0.1% SDS at 42°C for 15 min. The filters were
then exposed and autoradiographed with Kodak
X-ray film using an intensifying screen (Lightning Plus, DuPont Cronex, Wilmington, DE) at
–70°C.
Measurement of Intracellular Calcium
The relative intracellular calcium concentration was determined by F-4500 Fluorescence
Spectrophotometer (Hitachi, Japan). The confluent monolayers of A. albopictus cells (2 × 106
cells) were resuspended by mild digestion trypsin
and were incubated in 5 mM HEPES buffered-DMEM medium with a Ca2+ indicator fura-2/AM,
acetoxymethyl ester (3 µg/ml, Molecular Probes,
Eugene, OR) for 1 h at room temperature. The
cells were washed twice with PBS and further
incubated for 30 min in 0.5 ml of PBS to allow
deesterification of the indicator. The calibration
of 340/380 nm fluorescence ratio values to intracellular Ca2+ concentration, and 510 nm for emission filter followed the procedure of Grynkiewicz
et al. (1985). The results are expressed as the average standard error of values obtained for triplicate samples.
RESULTS
Effect of Depletion of Extracellular Calcium for
a Short Period and Ca2+-Restoration on Protein
Synthesis in Mosquito Cells
To determine whether the extracellular calcium concentration would affect the protein synthesis in A. albopictus cells in vitro, confluent
monolayers (2 × 106 cells) were incubated for 20
or 40 min with minimal Eagle medium (MEM)
containing 0.5 mM EGTA (low calcium medium)
or in the absence of EGTA with 0.5 mM of calcium (normal calcium medium) (Fig. 1). It was
observed that in cells incubated in low calcium
medium for 20 or 40 min, protein synthesis decreased (Fig. 1, lanes 2 and 3). However, in cells
incubated in low calcium medium, then returned
to normal calcium medium for 20 or 40 min, protein synthesis was similar to that in control cells
(Fig. 1, lanes 4 and 5, respectively). These results
show that the extracellular calcium concentration
can affect the protein synthesis profile. However,
this effect could be reverted when the cells were
Calcium in Aedes albopictus Cells
51
Effect of Prolonged Extracellular Ca2+-Depletion
on Recovery of Protein Synthesis Following
Ca2+-Restoration
Fig. 1. Effect of depletion of extracellular Ca2+ and its restoration on protein synthesis in A. albopictus cells cultured
for up to 40 min. A. albopictus cells were maintained in Ca2+free MEM medium in the presence of 0.5 mM EGTA (low
calcium medium) or without EGTA but with 0.5 mM of calcium (normal calcium medium). Lane 1 represents protein
synthesis profile of control cells. Lanes 2 and 3 represent
cells maintained in low calcium concentration for 20 and 40
min, respectively. Lanes 4 and 5 represent cells pre-incubated 20 min in low calcium concentration and returned to
a medium with normal calcium concentration for 20 and 40
min, respectively. The same amount of cells (2.25 × 105 cells)
was pulse labeled with [35S]-methionine (15 µCi/ml) for the
last 20 min in each experiment. The protein synthesis profile was determined by autoradiography of SDS-PAGE gels.
Molecular weight markers are indicated on the left.
returned to the normal calcium conditions. The
inhibition observed in these experiments may not
be due to the presence of EGTA, because cells incubated with EGTA and an excess of calcium ion
did not show this effect (data not shown).
In the results presented in Figure 1, it was
observed that the inhibition of protein synthesis
related to the decrease in calcium concentration
was a reversible effect. We investigated if this process could also be observed in the cells incubated
in low calcium medium for a longer period.
The results presented in Figure 2 show that
the cells incubated for 3 h in low calcium medium (Fig. 2A, lane 2) exhibited a drastic reduction in protein synthesis. However, when these
cells were returned to control medium for 20 or
40 min (Fig. 2A, lanes 3 and 4, respectively), the
cells recovered and resumed normal protein synthesis. Under these conditions, two proteins of
approximate molecular mass of 70,000 and 80,000
daltons were observed, as indicated in Figure 2A.
Densitometric analysis of the autoradiogram of
Figure 2A (lanes 2 and 3), showed that the total
protein synthesis increased approximately 6.4-fold
when the cells maintained 3 h in low calcium concentration (Fig. 2B, a) were returned to calcium
medium for 20 min (Fig. 2B, b).
Because the extracellular calcium depletion
seems to be a stress situation and as the proteins induced under this condition had molecular
weights similar to heat shock proteins (HSPs),
we investigated whether the HSP genes were being transcribed. For this purpose, when cells were
placed in Ca 2+ depleted medium, we utilized
Northern blot analysis to identify the HSP transcripts. The total cellular RNAs were extracted
from 2 × 106 cells, and 10 µg of RNAs was hybridized with a molecular probe containing
hsp70 cDNA (heat shock protein 70,000 dalton)
(Fig. 2C). The presence of hsp70 mRNA was detected in cells maintained for 3 h in low calcium
medium (Fig. 2C, lane 2) and in cells maintained
in this condition and then returned for 20 min to
control medium (Fig. 2C, lane 3). RNA from control cells, cultured in normal medium, did not
contain hsp70 mRNA (Fig. 2C, lane 1). The
hsp70 probe hybridized to 2.5 kilobases (kb)
mRNA as in our previous studies on A. albopictus cells (Carvalho and Fournier, 1991). In
contrast to hsp70, hybridization with the hsp80
probe (heat shock protein of 80,000 daltons) did
not detect any distinct band, thus indicating
52
Kawamura and Carvalho
Fig. 2. Effect of depletion of extracellular calcium and its
restoration on protein synthesis in cells maintained in low
calcium concentration for 3 h. A: Autoradiogram of protein
synthesis in control cells (lane 1), in cells maintained in
low calcium medium for 3 h (lane 2), or cells returned to
control medium for 20 and 40 min after 3 h in low Ca2+
medium (lanes 3 and 4, respectively). The molecular weight
markers are to the left of the autoradiogram and the 70,000
and 80,000 daltons induced proteins are on the right. B:
Densitometry of cells maintained in low calcium concentra-
tion medium for 3 h (a) and cells maintained in this condition and returned for 20 min to normal medium (b), corresponding lanes 2 and 3 from the autoradiogram of Figure
2A, respectively. C: Northern blot analysis for hsp70. The
total cellular RNA was probed for hsp70 cDNA. C shows the
Northern blot autoradiogram of control cells (lane 1), cells
maintained for 3 h in low calcium concentration (lane 2)
and cells maintained in this condition and returned for 20
min to normal calcium concentration (lane 3). The estimated
size of mRNA is indicated on the right size.
that the induced proteins in the 80,000 dalton
gel band does not belong to the HSPs family of
proteins (data not shown).
analysis by trypan blue showed that the cells conditioned as described above remained viable (data
not shown).
Effect of Extracellular Calcium Concentration
on Intracellular Calcium Concentration in A.
albopictus Cells
DISCUSSION
Finally, we determined if the extracellular
calcium concentration variation was able to
change the intracellular calcium concentration in
A. albopictus cells. For this analysis, we utilized
the Ca2+ fluorescent indicator Fura-2. A decrease
of 48% in the relative intracellular calcium concentration was observed in cells maintained for
3 h in low calcium concentration when compared
to cells maintained in normal medium (Fig. 3,
panels B and A, respectively). Dye exclusion
The calcium ion has an important regulatory role in diverse biological processes such as
the growth of cultured cells, signal transduction
(Carafoli, 1987) and coordination of a variety of
enzymes (Berridge, 1991). Recently, the importance of this ion either in regulation of protein
synthesis and expression of specific genes was
demonstrated in several mammalian cells (Brostrom et al., 1984; Gilchrist et al., 1994; Isogai
and Yamaguchi, 1995).
Results reported here showed for the first
Calcium in Aedes albopictus Cells
53
Fig. 3. Effect of low extracellular calcium on intracellular
calcium concentration in A. albopictus cells. Intracellular
calcium concentration of control cells (A) and cells main-
tained 3 h in low calcium concentration (B). The cells were
incubated during the 3rd h with fura-2 (3 µg/ml). Quantification was as described in Materials and Methods.
time the effects of changes in calcium concentration on protein synthesis in mosquito cells. In our
study, we showed that incubation of A. albopictus
cells in low calcium medium inhibited cellular protein synthesis (Figs. 1, 2A,B). This inhibition
could be reversed when the normal calcium concentration in the medium was restored (Figs. 1,
2A,B). These results are in agreement with observations on several types of vertebrate cells, in
which depletion of intracellular Ca2+ inhibited the
normal cellular mRNA transcription, and that
this process is reversible with restoration of calcium levels (Brostrom et al., 1983). The inhibition observed in our experiments may not be due
to the presence of EGTA, because cells incubated
with this chelator in the presence of calcium ion
did not show this effect (data not shown). This
observation was in agreement with Brostrom et
al. (1983) who verified that in mammalian cells,
addition of 1 mM Ca2+ in excess of the chelator
restored the rate of protein synthesis to that
nondepletion control preparations.
One question that may be related to our
results is whether protein synthesis inhibition
by EGTA was due to the interference in calcium
concentration or due to any other cation present
in the medium. Previous studies on this subject showed that Ca2+ seems to be the only cation that is critical for protein synthesis, since
it was the only cation that, when restored, led
to the protein synthesis recovery (Brostrom et
al., 1983).
We also observed the induction of two specific
proteins whose molecular mass is approximately
70,000 and 80,000 daltons in cells maintained for
3 h in low calcium concentration (Fig. 2A). Northern blot analysis of total RNA from control and
Ca 2+ -depleted cells showed the presence of
hsp70 mRNA only in Ca2+-depleted cells (Fig.
2C). This result suggests that the low calcium
concentration induced hsp70 mRNA. The apparent induction of hsp70 mRNA in A. albopictus
cells cultured in low calcium concentration is
in apparent contradiction with the data of Price
and Calderwood (1991), who suggested the dependence of the hsp70 gene expression on Ca2+
in several cell lines. However, these data are
controversial, because in some cell lines Hsp70
seems to be independent of calcium ion (Kim
and Lee, 1986). According to our results (Fig.
54
Kawamura and Carvalho
2A and C, lanes 2), calcium is not necessary to
the activation of the heat shock gene expression in A. albopictus mosquito cells.
The hsp70 mRNA induction by calcium
depletion has not been reported previously; however, Brostrom et al. (1983) observed that, in C-6
calcium-depleted cells, one unidentified 70,000
dalton protein seemed to be differentially influenced in its synthesis by calcium deprivation.
Northern blot analysis (Fig. 2C) suggests that the
70,000 dalton protein observed in our experiments
could be a member of Hsp70 family.
Northern blot analysis has not indicated the
presence of hsp80 mRNA in mosquito cells. Thus,
the 80,000 dalton protein observed in our studies
may belong to the group of glucose-regulated proteins (GRPs), denominated GRP78 and GRP94.
In mammalian cells, it was observed that the
depletion of intracellular calcium stores by calcium ionophore A23187 or thapsigargin, which
specifically inhibits the endoplasmic reticulum
Ca2+-ATPase, enhances the grp78 transcription
(Drummond et al., 1987; Li et al., 1994). However, we still have to determine if the 80,000
dalton protein observed in our experiments belongs to GRPs family.
When cells were maintained in low extracellular calcium concentration, intracellular concentration of Ca2+ ion decreased (Fig. 3). However,
when the cells were returned to the normal medium, intracellular calcium concentration returned
to normal levels. These results indicate a direct
relationship between extracellular and intracellular calcium concentration in mosquito cells.
Studies on vertebrate cells suggest that endoplasmic reticulum calcium homeostasis plays
a key role in the control of protein synthesis. It
has been concluded that disturbances in the endoplasmic reticulum homeostasis may contribute
to the suppression of protein synthesis triggered
by a severe metabolic stress (Doutheil et al.,
1997). This inhibition seems to be dependent on
activation of the double-stranded RNA-dependent
protein kinase (PKR). The calcium depletion from
the endoplasmic reticulum activates PKR, resulting in phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF-2α), which is critical
for protein synthesis initiation (Srivastava et al.,
1995; Alcazar et al., 1997). However, it still remains to be determined if the protein synthesis
inhibition observed in our experiments is related
to the eIF-2α phosphorylation.
ACKNOWLEDGMENTS
We are indebted to Mr. Marcelo Soares da
Mota e Silva and Mr. Paulo Sergio Lopes for excellent technical assistance.
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