BBRC Biochemical and Biophysical Research Communications 307 (2003) 503–509 www.elsevier.com/locate/ybbrc Tissue- and stressor-specific differential expression of two hsc70 genes in carp  brah aszl o Dorgai,b Magdolna A am,a and Edit Hermesza,* Khaled Said Ali,a L b a Department of Biochemistry, Faculty of Science, University of Szeged, P.O. Box 533, H-6701 Szeged, Hungary Bay Zolt an Foundation for Applied Research, Institute for Biotechnology, Derkovits fasor 2, H-6726 Szeged, Hungary Received 9 May 2003 Abstract Two genes expressing 70 kDa heat shock proteins were identified in Cyprinus carpio. The sequence similarities and the introninterrupted structure of the coding regions indicate that carp Hsc70-1 and Hsc70-2 belong to the Hsp70 cognate subfamily. The expressions of the two hsc70 genes were followed by semi-quantitative RT-PCR. Both genes are expressed under unstressed conditions in a characteristic tissue-specific manner. Inducibility of the response to elevated temperature, cold shock, and Cd treatment was investigated in the liver and muscle, in whole-animal experiments. Both genes were insensitive to or only weakly induced by the stressors, with two exceptions: Cd treatment resulted in an 11–13-fold enhanced induction of hsc70-1 in the liver and cold shock enhanced induction of hsc70-2 in the muscle by 7.5–10-fold. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Carp; Cold shock; Cd treatment; Differential expression; Hsc70; Teleostei A sudden temperature upshift and other types of environmental stresses induce the synthesis of a specific set of proteins: heat shock or stress proteins (Hsps). This response, highly conserved throughout evolution, is found universally from bacteria through lower eukaryotes to human. The Hsps themselves and their genes, among the best conserved phylogenetically, comprise several classes, one of them is the Hsp70 family, containing highly conserved and widely studied proteins with a molecular mass of about 70 kDa. Hsp70s play essential roles in protein metabolism under normal and stress conditions, e.g., de novo protein folding, membrane translocation, formation, and disassembly of protein complexes, or degradation of misfolded proteins (for reviews, see [1–3]). They consist of two domains: the 44 kDa N-terminal domain binds and hydrolyses ATP, while the more variable 30 kDa C-terminal interacts with unfolded polypeptides [4]. Hsp70s also interact with a number of other proteins, promoting specific chaperoning functions. Their expression is regulated by * Corresponding author. Fax: +36-62-54-48-87. E-mail address: hermesz@bio.u-szeged.hu (E. Hermesz). 0006-291X/03/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0006-291X(03)01206-3 environmental and physiological stresses and nonstressful conditions such as cell growth and development [5,6]. Some family members are at best weakly expressed under normal conditions and are inducible by heat and other stresses, allowing cells to cope with acute stressor insults (bona fide Hsp70s). Others (Hsp70 cognates, Hsc70s), expressed constitutively, are at best only slightly inducible, and play essential roles in the protein metabolism under normal conditions [1–3]. The highly related Hsp70 and Hsc70 are often suggested to have similar physiological functions [7]. Mammals appear to contain more than one isoform for both Hsp70 and Hsc70. There is a higher similarity between the members of the two subfamilies from different species than between Hsp70s and Hsc70s from the same species, e.g., hsc70 gene products from human, rat, and hamster are 99% similar, while human Hsp70 and Hsc70 amino acid sequences share only 85% identity. In fish, Danio rerio is the only example of variation in isoforms of Hsc70. The two zebrafish Hsc70s share 94% identity. During embryogenesis, the strong expression of one of them was found in the developing central nervous system and the differentiating somites, suggesting some 504 K.S. Ali et al. / Biochemical and Biophysical Research Communications 307 (2003) 503–509 specialized function during development [6,8]. In unstressed Rivulus marmoratus, the only identified Hsc70 exhibits tissue-specific expression [9]. This study reports the identification of two hsc70 genes in Cyprinus carpio. The encoded proteins are the first examples in lower vertebrates of Hsc70 isoforms with substantially lower identities than their counterparts in zebrafish or mammals. The expression patterns of the two hsc70s were compared in several tissues under unstressed conditions and in response to elevated temperature, cold shock, and Cd treatment in whole-animal experiments. The hsc70 expression is demonstrated to be regulated in an isoform- and tissue-specific and stressordependent manner. Materials and methods Animals and treatments. Carp (Cyprinus carpio) weighing 800– 1000 g were kept as described earlier [10]. In heat shock treatments, fish were transferred from 12 to 26, 28, or 30 °C, for up to 3 h, and in cold shock experiments to 5 °C for 1–5 h. Samples were taken from the tissues either immediately after the heat or cold treatment, or after a 1-h recovery at the acclimatization temperature. For treatment with Cd, the carp were transferred into 100-liter water tanks containing 10 mg/l Cd (cadmium acetate, Fluka) for up to 96 h, under static conditions. Tissues were frozen immediately in liquid nitrogen and stored at )80 °C. Nucleic acid preparation. The procedure for RNA extraction and cDNA synthesis was described earlier [10]. Genomic DNA was extracted from carp liver by a modified version of the salting-out procedure [11]. Briefly, approximately 10 mg of frozen tissue was incubated in 0.5 ml of lysis buffer (10 mM Tris–HCl, pH 8.2, 5 mM EDTA, 0.2% SDS, and 200 lg/ml protease K) at 55 °C overnight. 150 ll of saturated NaCl was added, and the mixture was shaken vigorously and centrifuged at 3000 rpm for 15 min. DNA was precipitated with 0.75 volume of isopropanol, transferred into 75% ethanol, incubated overnight, collected by centrifugation, dried under vacuum, dissolved in H2 O, and stored at )80 °C. PCR amplification. 50 ng of genomic DNA or 2 ll of RT reaction product was added to 48 ll of a PCR mixture containing 250 lM of each dNTP, 50 pmol of primers, 1
Sigma PCR buffer/MgCl2 , and 5 U of Taq polymerase (Sigma). Amplification was performed in a PTC 200 Peltier Thermal Cycler (MJ Research), using 5 cycles of 95 °C for 30 s, 45 °C for 30 s, and 72 °C for 90 s, followed by 30 cycles with an annealing temperature of 55 °C. The amplified products were separated on 1–1.8% agarose gels (Sigma), isolated from them via Ultrafree-MC Centrifugal Filter Units (Millipore), inserted into pGEM-T Easy vector (Promega), and transformed into Escherichia coli DH5a cells. The recombinant clones were screened for and the insert sequence was determined. For semi-quantitative measurements, primers 7F/8R for hsc70-1 and 9F/10R for hsc70-2 were used at an annealing temperature of 60 °C in all cycles. The number of amplification cycles, during which PCR product formation was limited by the template concentration, was determined in pilot experiments: for b-actin 25 and for hsc70s 30 cycles were used. Northern blot analysis. Fractionation of RNA samples, blotting, and hybridization were performed as previously [10]. DNA probes (isolated 289 bp hsc70-1 and 234 bp hsc70-2 cDNA fragments, see Fig. 1A) were labeled with [a-32 P]dCTP by random priming using a High Prime DNA Labeling Kit (Boehringer–Mannheim). Approximately equal RNA gel loading was determined by visualization of the ribosomal RNA bands, after staining with ethidium bromide. Primers. The design of PCR primers for evolutionally conserved regions was based on multiple alignments of sequences from zebrafish (GI:17061841 and GI:1865782), trout (GI:17129570 and GI:246719), Xiphophorus maculatus (GI:17061837 and GI:17061839), xenopus (GI:64796 and GI:1326171), chicken (GI:211941 and GI:2996407), mouse (GI:193983 and GI:309319), and human (GI:386785 and GI:13273304). Oligonucleotides used in this work are listed in the table below. hsp70/hsc70-specific primers: 1F: 50 -GCTGTTGGCATTGACCTGGG-30 2R: 50 -TCTGGGTTAATGCTCTTGTT-30 4R: 50 -GGTGATGGTGATCTTGTTCT-30 6R: 50 -GTCAACCTCCTCAATGGTTG-30 Carp hsc70-2-specific primers (AY219844): 3-2F: 50 -CTGGCTTGAATGTTCTGGGT-30 5-2F: 50 -CCAAGACTACTTCAACGGCAA-30 9F: 50 -AAGAATGGTCTGGAATCCTAT-30 10R: 50 -GCCTCCAGCACTCTGGTACAG-30 Carp hsc70-1-specific primers (AY120893): 3-1F: 50 -CTGGCCTTGACGTCCTCCGC-30 5-1F: 50 -TCAGGACTTATTTAACGGCC-30 7F: 50 -ATCGACCTGGGCACCACCT-30 8R: 50 -CTTCCATCTTGGGCTTACT-30 G-1R: 50 -TTGGCTCGTTGGTAATGCGG-30 G-2F: 50 -TATATGAGGGAGAGAGAG-30 b-Actin-specific primers (M24113): b-3: 50 -GCAAGAGAGGTATCCTGACC-30 b-4: 50 -CCCTCGTAGATGGGCACAGT-30 Measurement of hsc70 mRNA levels. At each experimental time point, three to four fish were used to prepare RNA. RT-PCRs for each animal were performed in triplicate to increase the reliability of the measurements. For normalization of the hsc70 mRNA amount, the carp b-actin mRNA level was used as an internal standard. Images of autoradiograms and ethidium bromide-stained agarose gels were digitized with a GDS 7500 Gel Documentation System and analyzed with GelBase/GelBlot Pro Gel Analysis Software (UVP). The relative levels of hsc70 mRNAs are expressed as ratios [hsc70/b-actin]
100. The results were submitted to Student’s t test analysis, with a probability P < 0:05 taken as the limit of significance. Analysis of Cd content. Tissues were dried and digested with HNO3 / H2 O2 . The Cd contents of the homogenates were determined with a Hitachi Z8200 Zeeman polarized atomic absorption spectrophotometer. Flame or graphite furnace atomization was used, depending on the Cd concentration. Cd contents are reported in lg/g dry weight. Results Identification of two hsc70 genes in carp In order to design PCR primers for amplification, all Hsp70 and Hsc70 database entries corresponding to both inducible and constitutive forms from the same fish species were selected, together with other pairs from evolutionally distant species. Entries representing only one of the two forms from a given species were not included. The sequences were aligned, and regions conserved in both the Hsp70 and Hsc70 primary structures were sought, and ordered further on the basis of the highest similarity at a nucleic acid level. As a result, four K.S. Ali et al. / Biochemical and Biophysical Research Communications 307 (2003) 503–509 505 Fig. 1. (A) Alignment of carp Hsc70-1 and Hsc70-2 amino acid sequences with their closest relatives. Hsc70-1 is taken for reference; in the other sequences, only the differences are indicated. Dots represent identities; dashes stand for gaps. The positions of the oligonucleotides used in this work are underlined and their names are given above the Hsc70-1 sequence. Vertical arrows point to the positions of the introns in hsc70-1. The probes for Northern blot hybridizations were amplified with the primer pairs 7F/8R for hsc70-1 and 9F/10R for hsc70-2. (B) Schematic structure of carp hsc70-1. Black boxes denote exons and thin lines denote introns. The intron sizes are indicated. 506 K.S. Ali et al. / Biochemical and Biophysical Research Communications 307 (2003) 503–509 regions were selected for primer design, two at the ends of the amino- and carboxyl-terminal regions and the other two located internally (primers 1F, 2R, 4R, and 6R, Fig. 1A). The first oligonucleotide pair used (primers 1F and 2R) had the potential to amplify the N-terminal coding segments from both hsp70 and hsc70 mRNAs. However, the basal expression of the newly identified carp hsp70 gene is below the level of detectability by RTPCR in the brain and muscle [12]. mRNA prepared from muscle was therefore used as a template and productive amplification was expected only from hsc70specific template(s). We used an annealing temperature of 45 °C during the first 5 cycles, in order to allow efficient priming from the carp template(s), conceivably containing mismatches to the evolutionally conserved regions used for primer synthesis. The major RT-PCR product had the size of 1 kb expected for amplification from hsc70-specific template(s). This product was inserted into a cloning vector and the sequence of the inserts was determined. The analysis of nine independent sequences revealed two cDNA species, one represented by seven and the other by two clones. On the basis of the determined sequences, two gene-specific primers were synthesized (3-1F and 3-2F) and overlapping coding region segments were amplified with primer pairs 3-1F/4R and 3-2F/4R, respectively. Cloning and sequencing of the products were performed as above. The whole procedure was repeated with two additional gene-specific primers 5-1F and 5-2F paired with primer 6R. Thus, 2 cDNA species could be unambiguously compiled from each set of three overlapping amplicons, which we termed carp hsc70-1 and hsc70-2. Both sequences contained an open reading frame spanning the entire length (Fig. 1A). Database searches indicated that the closest relatives of the coding regions and the deduced proteins were the known hsc70 and hsp70 cDNAs and proteins. Carp hsc70-1 was most similar to hsc70 described from Rivulus marmoratus (81% and 93% at DNA and at protein levels), while carp hsc70-2 exhibited highest homology to hsc70 from zebrafish (91% and 96%, respectively). Phylogenetic analysis also placed the two carp Hsc70 sequences in a diverse group containing all Hsc70s from fish, well separated from a compact, second group populated by the fish Hsp70s (Fig. 2). Additional evidence for classification of the newly identified two carp genes as hsc70s could be the presence of introns in the coding regions. Hence, we amplified and sequenced three segments of the carp hsc70-1 gene by specific primer pairs (1F/G-1R: 1682 bp; 3-1F/4R: 1668 bp; and G-2F/6R: 1121 bp; GenBank No. AY219845). The genomic and cDNA sequences were identical in the overlapping segments, indicating the specificity of the amplification from the hsc70-1 gene. Comparison of the genomic and cDNA Fig. 2. Phylogenetic analysis of fish Hsp70 and Hsc70 sequences. Fulllength sequences were retrieved from GenBank and aligned with the aid of CLUSTAL W [26]. The phylogram was generated by the PHYLIP package [27] and viewed by TreeView [28]. For all analyses, the public facility of the Institut Pasteur was used (www.pasteur.fr). sequences revealed seven introns within the coding region, at exactly the conserved positions characteristic for eukaryotic hsc70s (Fig. 1B) [9]. Carp hsc70-2 was not characterized as extensively as hsc70-1, but a similar amplification from genomic DNA template indicated intron 7 also present in hsc70-2. Basal expression of carp hsc70s In order to characterize the heat shock genes further, we determined their basal expression levels in the muscle, heart, brain, liver, and kidney, by measuring the amount of gene-specific mRNA relative to b-actin (Fig. 3A). The specificity of the gene-specific primer pairs used for semiquantitative RT-PCR measurements was first tested on cloned hsc70 templates: no cross-priming was found. The highest hsc70-1 mRNA level was found in the muscle, comparable to that of b-actin. About 25% of this level was measured in the brain and heart, while the expression was virtually undetectable in the liver and kidney. The tissue-specific expression pattern of hsc70-2 differed markedly, with the highest level in the kidney (55–60% of b-actin mRNA), slightly less in the brain and liver (45% and 40%), and even less in the heart (20%). The muscle, which contained the highest amount of hsc70-1 mRNA, displayed the lowest hsc70-2-specific message level (0–15%, depending on the animal). The basal expression levels of the hsc70s were tested independently by Northern hybridization in the muscle and liver, the organs selected for induction studies. The result agreed well with that of RT-PCR measurements: hsc70-1 mRNA was readily detected in the muscle, but not in the liver, while the hsc70-2 probe detected about 4.5-fold more mRNA in the liver than in the muscle (Fig. 3B). The almost identical length of the two coding regions does not offer an explanation for the size dif- K.S. Ali et al. / Biochemical and Biophysical Research Communications 307 (2003) 503–509 507 Fig. 3. Expression of carp hsc70-1 and hsc70-2 in various organs of unstressed animals. (A) Representative RT-PCR amplification. RNAs for template were prepared from the brain (B), heart (H), muscle (M), liver (L), and kidney (K). (B) Northern blot hybridization with hsc70-1 (upper left panel) and hsc70-2 (upper right panel) specific probes. Ribosomal RNAs were visualized by ethidium bromide staining for loading control (bottom panel). ference of the mRNAs determined by this analysis (3.2 kb for hsc70-1 and 2.9 kb for hsc70-2); divergences in the 50 and/or 30 non-coding regions of the transcripts must therefore be assumed. Inducibility of carp hsc70-1 and hsc70-2 The constitutive nature of the hsc70s was tested by heat and cold shock, and by treatment with the aspecific stressor Cd. For this analysis, we selected the muscle and the liver, in which the ratios of the basal expressions of hsc70-1 and hsc70-2 differed markedly. A 14 °C jump in temperature did not result in a significant increase in hsc70-1 expression in the muscle after either a 30-min or a 3-h incubation. However, a further 1-h of recovery at the adaptation temperature led to a modest, but measurable induction: 1.5-fold and 2–2.5fold, respectively. The inducibility of hsc70-2 by heat was significantly higher in the muscle: a 3-fold increase in specific mRNA level was measured after a 30-min heat shock, with the highest induction (4–4.5-fold) after a 3-h incubation followed by a 1-h recovery (Fig. 4). In the liver, both genes proved insensitive to heat shock. The hsc70-1 mRNA level was not detected at any treatment time point, while the induction of hsc70-2 was increased by <2-fold (data not shown). Further induction of temperature increases (16 and 18 °C jumps) did not change the pattern or level of inducibility of either gene in either organ (data not shown). To investigate the effect of cold shock, the animals were exposed to a 7 °C drop in temperature, with or without their return to the adaptation temperature for 1 h. In the muscle, the expression of hsc70-1 was downregulated; the amount of specific mRNA was 50% of the control value after a 2-h cold shock followed by a 1-h recovery, and not significantly different from this at 5 þ 1 h. Cold shock resulted in an opposite effect on the expression of hsc70-2: a 7.5–10-fold induction in the muscle after 2 þ 1 h of treatment, which was not reduced significantly after 5 þ 1 h (Fig. 5), and a much Fig. 4. Induction of carp hsc70s by heat shock in the muscle. The values are the averages ( SD) of at least three independent measurements and are calculated relative to the control values. Light shading denotes hsc70-1; dark shading denotes hsc70-2. Fig. 5. Effects of cold shock on expression of carp hsc70s in the muscle. Animals were transferred from 12 to 5 °C, exposed to this temperature for the time indicated on the top, and transferred back to 12 °C for 1 h. The apparent downregulation of hsc70-2 at 1 þ 1 h is due to individual variation; the basal level of hsc70-2 varies between undetectable and 15%. The averages of independent experiments do not show significant changes by 1 þ 1 h of treatment. smaller (1.5-fold) induction in the liver, in which no hsc70-1 mRNA was detected at any time point (data not shown). The expressions of hsc70-1 and hsc70-2 were also investigated by the incubation of animals in the presence of 10 mg/l Cd. No induction of either gene was detected in the muscle at any time point. In the liver, both were induced. The hsc70-2 mRNA level increase was 508 K.S. Ali et al. / Biochemical and Biophysical Research Communications 307 (2003) 503–509 Fig. 6. Induction of carp hsc70s by Cd treatment in the liver. (A) Representative RT-PCR amplification. RNAs were prepared after exposure to 10 mg/l Cd at the time indicated on the top. (B) Kinetics of induction. For calculation of the fold induction see the legend to Fig. 4. Light- and dark-shaded columns indicate hsc70-1 and hsc70-2, respectively. Triangles show Cd accumulation. relatively modest (2–3-fold). In contrast, the expression of hsc70-1 was strongly elevated: in experiments where a detectable expression was found in the controls, an 11– 13-fold induction was measured by 96 h; and in experiments where no control expression was experienced, the induction between 24 and 96 h was 7-fold (Fig. 6). This organ-specific induction correlated well with the Cd content of the tissues: there was no measurable Cd accumulation in the muscle at any time point, while in the liver the Cd content reached 30 lg/gdw by 96 h (Fig. 6B). Discussion We have identified two genes, coding for Hsc70-1 and Hsc70-2 in common carp. Sequence similarities, the intron-interrupted structures of the genes, and the results of phylogenetic analysis suggest that these proteins are representatives of the 70 kDa heat shock cognate chaperones. The expressions of the carp hsc70 genes under unstressed conditions are consistent with their cognate nature: the basal expression levels (in organs where the genes are expressed) are substantial and even comparable to that of the b-actin gene. Also consistent with the classification is the relative insensitivity of both carp hsc70s to heat shock. Additionally, both genes were insensitive to cold shock in the liver and to the heavy metal Cd in the muscle. There are a number of examples of more than one Hsc70 being described from the same organism. However, only two lower vertebrates, D. rerio [6,8] and Xenopus laevis [13,14], are known to express two or more closely related Hsc70s (94% and 94–97% identities, respectively). Carp Hsc70-1 and Hsc70-2 are more similar to their closest homologs from other species than to each other and are the first examples of Hsc70s from the same fish that differ substantially (88% at a protein level and 78% at a DNA level). Known hsc70s exhibit widely divergent expression patterns: hsc70 in the mouse is expressed in all organs in a constitutive manner, with some quantitative differences between the tissues [15], while hsc70t is mainly expressed in male germ cells [16]. hsc70 in the zebrafish and mouse is spatially and temporarily regulated in the developing embryo [6,17]. The characteristic basal expression pattern of the carp hsc70 genes is unique: there are organs where both specific mRNAs were detected, and others where one is expressed predominantly, in a “complementary” manner. In this sense, their expression patterns lie between the two extreme examples above. The carp hsc70s were relatively insensitive to the stressors applied, except in two organs. A 10–12-fold induction of hsc70-1 by Cd was measured in the liver. Cd exposure did not cause a substantial induction of the expression of hsc70 in various studies [18–21]. Accordingly, carp hsc70-1 is the first example of this subfamily to be known to have such a capacity of reacting to Cd treatment. Similarly, the 7.5–10-fold induction of carp hsc70-2 by cold shock in the muscle is not paralleled by many examples. In few cases when the effects of cold shock were investigated, no induction of hsc70 s was observed; rather, the inducibility was characteristic of hsp70 [22,23]. It has been suggested that the Hsc70s from the same species have distinguishable functions, best exampled by the roles of the yeast Hsc70 homologs Ssc1p and BiP in post-translational protein translocation [24]. Our results support this idea. The “compleplementary” nature of the basal hsc70-1 and hsc70-2 expressions in the carp liver, muscle, and kidney, and the differential expression in response to two stressors, indicate differences in regulation, which in turn suggests some specialization in function. 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