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