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Cloning and characterization of the hsp70 multigene family from silver sea bream: Modulated gene expression between warm and cold temperature acclimation
BBRC Biochemical and Biophysical Research Communications 330 (2005) 776–783 www.elsevier.com/locate/ybbrc
Cloning and characterization of the hsp70 multigene family from silver sea bream: Modulated gene expression between warm and cold temperature acclimation Eddie E. Deane, Norman Y.S. Woo * Department of Biology, The Chinese University of Hong Kong, Shatin, NT, Hong Kong SAR, China Received 7 March 2005 Available online 17 March 2005
Abstract The genes encoding heat shock cognate 70 (hsc70) and inducible heat shock protein 70 (hsp70) were cloned and characterized from silver sea bream liver. Upon acute heat shock (+7 C), the transcript abundance of hsc70 was increased 1.7-fold whereas the transcript abundance of hsp70 increased 6.7-fold. The chronic acclimation of sea bream to cold temperature (12 C) resulted in a downregulation of hsc70 and an upregulation of hsp70 in comparison to levels in sea bream kept at a warmer temperature (25 C). The expression of heat shock transcription factor I was also increased during cold temperature acclimation. Increased amounts of hepatic insulin-like growth factor 1 transcript, serum thyroxine (T4), and triiodothyronine (T3) were also found during cold temperature acclimation whereas serum cortisol remained unchanged. The results from this study demonstrate how temperature acclimation, in fish, can affect the regulation of the hsp70 multigene family and hormonal factors that are associated with anabolism. 2005 Elsevier Inc. All rights reserved. Keywords: Fish; Temperature; hsc70; hsp70; hsf1; IGF-1; Hormones
Disruption of normal cellular processes can cause the rapid and increased synthesis of a group of proteins belonging to the heat shock protein (HSP) families. Members of these different HSP families are grouped according to molecular size and perform varying and different roles in the cell. The HSP90 family is involved in steroid receptor formation and protein folding [1], the HSP70 family is necessary for translocation and protein folding [2], and HSP60 is involved in protein stability and folding [3,4]. These HSP families are important for immune function [5] and have been demonstrated to be upregulated in fish during stress [6]. Of all the HSP families, HSP70 has been most widely studied as a biomarker of stress [7] and the major inducing factor for HSP70 upregulation is the occurrence of damaged cellular pro-
*
Corresponding author. Fax: +852 26035646. E-mail address: [email protected] (N.Y.S. Woo).
0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.03.039
tein [8]. The HSP70 family is coded for by two different genes, a cognate or constitutive type (hsc70) and an inducible type (hsp70), and both of these genes encode proteins that play key roles in the cell as molecular chaperones [2]. In cells, hsc70 remains unchanged or slightly upregulated upon exposure to stress whereas hsp70 is highly induced, from low basal levels, with transcriptional control mediated via heat shock factor 1 (HSF1) that binds in a trimeric form to a hsp70 gene promoter [9]. As fish are aquatic ectotherms, they have to contend with continuous fluctuations in water temperature and the well-documented response is to modulate profiles of metabolic enzymes [10–12] and alterations in endocrine control are also known to occur [13,14]. Tissue HSP70 could be regulated according to external water temperature, but it has yet to be reported on how the gene expression of both hsc70 and hsp70 as well as hsf1 is adjusted in fish tissues in response to warm and cold water temperature acclimation. In the present study, we report on the
E.E. Deane, N.Y.S. Woo / Biochemical and Biophysical Research Communications 330 (2005) 776–783
cloning and characterization of both hsc70 and hsp70 from silver sea bream (Sparus sarba) liver. The gene expression profiles for hsc70, hsp70, and hsf1 from sea bream acclimated between cold water (12 C) and warm water (25 C) are presented. To correlate the heat shock protein response with aspects of growth physiology, gene expression studies for the mitogenic hormone insulin-like growth factor 1 (IGF-1) and serum levels of cortisol, thyroxine, and triiodothyronine are reported. To our knowledge this is the first study to clearly define molecular regulation of hsp70, hsc70, and hsf1 genes in fish acclimated to different water temperatures and demonstrates how the hsp70 multigene family is differentially expressed in fish kept at different temperatures.
lated and subjected to further rounds of screening. After three rounds of screening, 10 putative clones for each gene were selected and converted to plasmid by in vivo excision using Escherichia coli strain BM25.8 (Clontech). Plasmids were cycle sequenced using an ABI PRISM dye terminator kit and reaction products were analyzed on an ABI 310 Genetic Analyzer (Perkin Elmer, USA). RT-PCR analysis of hsc70, hsp70, hsf1, and IGF-1. First strand cDNA was synthesized from 1 lg of total RNA, from each sample, using a Powerscript reverse transcriptase kit (Clontech). First strand cDNA synthesis was allowed to proceed at 42 C for 1 h after which time the reaction was incubated at 70 C for 15 min. For PCR amplification of genes, from first strand cDNA template, oligonucleotide primers were designed from the nucleotide sequences of hsc70 and hsp70 cloned genes, and primers for hsf1 and IGF-1 have previously been used for studies on sea bream [18]. As a normalization control for each RT-PCR, primers specific for 18S rRNA were used [19]. All primers were synthesized by Genset (Singapore) and had the following sequences: 5 0 -ATCAGTGATGACGACAA-3 0 5 0 -TGACCCCCCCCCAGGGGC-3 0 hsp70: 5 0 -ATCAGTGAGGAGGACAAA-3 0 5 0 -CTGGGAGCCGCTTCCTGC-3 0 hsf1: 5 0 -CCCCAGTGGAACCAGCTTCCATG-3 0 5 0 -GGATGTTGGAATTCCGTGTCATC-3 0 IGF-1: 5 0 -AGTGCGATGTGCTGTATC-3 0 5 0 -CAGCTCACAGCTTTGGAA-3 0 18S: 5 0 -GCCAAGTAGCATATGCTTGTCTC-3 0 5 0 -AGACTTGCCTCCAATGGATCC-3 0
hsc70:
Materials and methods Experimental fish and holding conditions. Adult silver sea bream (Sparus sarba) weighing between 200 and 300 g were purchased from a local fish farm and transferred to 1000 L seawater tanks. The water in the tanks was at a temperature of 24–25 C, fully aerated, and fish were fed ad libitum once daily with a formulated diet [15]. Fish were allowed to acclimate to experimental conditions for three weeks prior to temperature exposure. Acute heat shock. Fish were divided into three groups (n = 5) and acclimated to opaque laboratory aquaria for two days prior to heat shock experiments. The seawater in the aquaria was changed daily and one group served as a control with water temperature maintained at 25 C. A second group of fish were subjected to a temperature increase of approximately 0.12 C/min (using an immersion heater) until 32 C was attained. Fish were maintained at this temperature for two hours, killed by spinal transection, and livers were removed. A third group of fish were also subjected to a stress of 32 C, for two hours, after which time the immersion heater was removed and the fish were allowed to undergo a recovery period of 24 h at 25 C prior to liver removal. Total RNA was extracted from the liver tissues using a Qiagen RNeasy mini kit (Qiagen), treated with DNase 1 (Gibco-BRL), quantified spectrophotometrically, and stored at 70 C. Temperature acclimation. Two groups of fish (n = 7 per group) were used for temperature acclimation studies. One group was maintained at 25 C and the second group of was kept at 12 C for one month. The water temperature in both of the tanks was maintained by using a system of water coolers and heaters. The water for each group was aerated and fish were fed ad libitum once daily with a formulated diet. After one month, blood was withdrawn from the caudal vessels, allowed to clot at room temperature for 1 h, and then centrifuged at 10,000g for 10 min to obtain serum. The serum was removed, aliqoutted, and stored at 70 C. After blood withdrawal, fish were killed by spinal transection, and livers were removed and extracted for total RNA as previously described. Cloning and characterization of hsc70 and hsp70 genes. A silver sea bream liver cDNA library was constructed using a SMART cDNA library construction kit (Clontech, USA) and 106 plaques of the library were transferred to Hybond NX plaque lifts (Amersham–Pharmacia Biotech, UK). Plaque lifts were prehybridized for 4 h at 60 C in Rapid-Hyb buffer (Amersham) and then hybridized at 65 C for 16 h with a [32P]dCTP-labeled putative hsc70 clone from sea bream [16] or a putative hsp70 DNA fragment isolated using specific PCR primers designed from the zebrafish hsp70 gene [17]. After hybridization, plaque lifts were washed twice in 2· SSC (1.8%w/v sodium chloride; 0.9%w/v trisodium citrate)/0.1% SDS for 30 min at 65 C and then once in 0.1% SSC/0.1% SDS for 30 min at 65 C. Membranes were exposed to X-ray film (Kodak, USA) and positive plaques were iso-
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(sense) (antisense) (sense) (antisense) (sense) (antisense) (sense) (antisense) (sense) (antisense)
PCRs (50 ll) containing 2 ll of first strand cDNA, 0.2 ll Taq DNA polymerase (Promega, USA; 5 U/ll), 5 ll MgCl2 (25 mM), 5 ll reaction buffer, 0.5 ll dNTP mix (10 mM), and 1 ll of each primer (50 lM) were used. PCR amplification was performed using a PTC-100 thermal cycler (MJ Research, USA) with cycle parameters of 94 C for 1 min, 55 C for 1 min, 72 C for 1 min, and a final extension of 72 C for 4 min. To ensure amplification was at the midpoint of the linear phase of amplification, for each gene, preliminary RT-PCRs were performed. A single PCR product, of expected size, was obtained for each gene of interest and these were subcloned into pCRscript plasmid vector (Stratagene, USA), followed by cycle sequencing to confirm identity. No PCR products were detected from negative controls (reactions without reverse transcriptase added). To confirm the specificity of each RT-PCR and to establish stringent hybridization conditions for subsequent analysis, an aliquot (10 ll) was taken from a number of representative samples and transferred to Hybond-N+ membrane (Amersham). Purified cDNA fragments of each gene were radiolabeled using a Rediprime random labeling kit (Amersham) and used as probes for membrane hybridization. Blots were hybridized at 55 C for 16 h, then washed twice with a 2· SSC/0.1% SDS solution for 30 min, once in 0.1· SSC/0.1% SDS at 65 C for 15 min, air-dried for 15 min, and then autoradiographed at 80 C. From preliminary hybridizations, it was established that probes were specific for corresponding amplified fragments from RT-PCRs and for semi-quantification of transcripts, samples were analyzed together in a single hybridization using DNA dot blots which were prepared using a Bio-Dot microfiltration manifold (Bio-Rad). To test for the linearity of detection during subsequent scanning procedures, PCR amplification products were diluted 5-, 10-, 50-, and 100-fold, prepared and blotted according to instructions supplied with Hybond-N+ membrane (Amersham). Membranes were hybridized and washed as described above and exposed to storage phosphor screens (Molecular Dynamics, USA) for 3 h at room temperature after which time the screens were scanned using the Storm PhosphorImaging system with ImageQuant software (Molecular Dynamics) for quantification of amplified fragment. The abundance of each specific gene fragment was normalized to the corresponding 18S abundance in all samples.
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Serum hormone analysis. Serum cortisol was assayed using a competitive ELISA kit (IBL, Hamburg) previously validated for use with sea bream samples [20]. Serum triiodothyronine (T3) and thyroxine (T4) were assayed using competitive ELISA kits (Biomerica, USA) that have been previously validated for use with sea bream samples [20]. Statistical analysis. Normalized transcript abundance, for each gene studied, were subjected to either a one way ANOVA to test for significance followed by a Student–Newman–Keuls test (Jandel Scientific) or a StudentÕs t test to delineate significance between groups. Significant differences were accepted if p < 0.05.
Results Isolation and characterization of hsc70 and hsp70 genes Using specific gene fragments, as radiolabeled probes against a liver cDNA library, 10 putative clones for hsc70 and hsp70 were isolated. The sea bream hsc70 open reading frame was 1950 bp in length coding for a protein of 649 amino acids (Fig. 1) whereas the sea
Fig. 1. Nucleotide and deduced amino acid sequence of silver sea bream heat shock cognate 70 (hsc70). The nucleotides are numbered on the left and the amino acids are numbered on the right. An asterisk above the last three nucleotides indicates a stop codon. The sequence encoding sea bream hsc70 has been deposited with the GenBank under Accession No. AY436786.
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bream hsp70 open reading frame was 1920 bp in length coding for a protein of 639 amino acids (Fig. 2). The amino acid homology between sea bream hsc70 and hsp70 was 85% which was to be expected since both genes belong to the same multigene family. After sequence comparisons using the Basic Local Alignment Search Tool Program [21], it was found that sea bream
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hsc70 shared 80–91% amino acid homology with many other animal hsc70 genes and similarly sea bream hsp70 shared a 77–86% amino acid homology with many other animal hsp70 genes. The sequences encoding sea bream hsc70 and hsp70 have been deposited with the GenBank under Accession Nos. AY436786 and AY436787, respectively.
Fig. 2. Nucleotide and deduced amino acid sequence of silver sea bream inducible heat shock protein 70 (hsp70). The nucleotides are numbered on the left and the amino acids are numbered on the right. An asterisk above the last three nucleotides indicates a stop codon. The sequence encoding sea bream hsp70 has been deposited with the GenBank under Accession No. AY436787.
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Expression analysis of hsc70 and hsp70 during acute heat shock The effects of an acute heat shock on hepatic hsc70 and hsp70 expression were studied, using an RT-PCR assay. The transcript abundance of hsc70 increased 1.7-fold following heat shock and then returned to similar levels as that found in non-heat-shocked fish, after a 24 h recovery period. The transcript abundance of hepatic hsp70 increased 6.7-fold following acute heat shock and also returned to similar levels as that found in non-heat-shocked fish, after a 24 h recovery period. These results are presented in Fig. 3.
(Fig. 4). Sea bream at 12 C also had 2.1-fold higher amounts of hsf1 transcript in comparison to fish acclimated to 25 C (Fig. 4). The levels of IGF-1 transcript were also found to be 2-fold higher in 12 C acclimated fish (Fig. 5).
Expression analysis of hsc70, hsp70, hsf1, and IGF-1 during warm and cold temperature acclimation Using an RT-PCR coupled with radioisotope probing approach, the transcript amounts of hsc70, hsp70, hsf1, and IGF-1 genes could be studied in liver tissue from sea bream acclimated to warm (25 C) or cold (12 C). The transcript abundance of hsc70 was found to be 2.3-fold higher in fish kept at 25 C in comparison to 12 C acclimated fish whereas the transcript abundance of hsp70 displayed an opposite trend with 2-fold higher levels at 12 C in comparison to fish at 25 C
Fig. 4. Expression of hepatic hsc70, hsp70, and hsf1 in sea bream acclimated to 12 and 25 C. RT-PCR was used to amplify transcripts and phosphorimaging analysis was used for transcript quantification. Data are presented as mean normalized transcript abundance (n = 7) ± SEM and an asterisk above the bar denotes a significantly different mean value (p < 0.05).
Fig. 3. Expression of hepatic hsc70 and hsp70 during acute heat shock of sea bream in vivo. Fish were kept at control (25 C) or subjected to an acute heat shock (HS) for 2 h at 32 C or subjected to a heat shock and then allowed to recover, at control temperature for 24 h (HS/R). Transcripts for all samples were quantified using phosphorimaging and the amount of hsc70 and hsp70 transcripts was normalized to the corresponding 18S transcript abundance of each sample and expressed in arbitrary units as mRNA abundance. All values are expressed as means ± SEM (n = 5) and different letters above a bar mean indicate values which are significantly different from each other (p < 0.05).
Fig. 5. Expression of hepatic IGF1 in sea bream acclimated to 12 and 25 C. RT-PCR was used to amplify transcripts and phosphorimaging analysis was used for transcript quantification. Data are presented as mean normalized transcript abundance (n = 7) ± SEM and an asterisk above the bar denotes a significantly different mean value (p < 0.05).
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Fig. 6. Measurements of serum cortisol (A), serum T4 (B), and T3 (C) in silver sea bream acclimated to 12 and 25 C. Serum cortisol is expressed as nanogram per milliliter and T4 and T3 are expressed as lg/100 ml. Data are presented as mean values (n = 7) ± SEM and an asterisk above a bar denotes a significantly higher mean value (p < 0.05).
Serum cortisol, T4, and T3 analysis The serum levels of cortisol remained unchanged between sea bream at 12 C (10.6 ± 1.2 ng/ml) and 25 C (11.0 ± 1.5 ng/ml). The amounts of serum T4 were 1.9fold higher in fish acclimated to 12 C (8.7 ± 1.4 lg/ 100 ml) than 25 C (4.6 ± 1.1 lg/100 ml) and similarly the amount of serum T3 was 1.8-fold higher in fish at 12 C (4.0 ± 0.31 lg/100 ml) than 25 C (2.2 ± 0.3 lg/ 100 ml). These data are presented in Fig. 6.
Discussion Presently only a relatively small number of genes from the hsp70 family in fish have been cloned and characterized (see [22] for review), and a clear delineation between constitutive and inducible members of the hsp70 family, in teleosts acclimated between warm and cold temperature, remains to be elucidated. To address this present void, both constitutive (hsc70) and inducible (hsp70) genes from sea bream liver were cloned and
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characterized in this study. The amino acid homology between hsc70 and hsp70 was high but analysis of the heterogeneous carboxyl regions of hsc70 and hsp70 in fish defines certain features that allowed us to differentiate between these two genes. The constitutive hsc70 genes are slightly longer than hsp70 genes and the carboxyl region of these genes is highly heterogeneous as hsc70 displays a series of unique repeat GGXP motifs and a signature nonapeptide SGPTIEEVD at the end [23–26]. In fish, inducible hsp70 genes, the GGXP motif occurs once in most cases and the last nine amino acids are not the same as the terminal hsc70 nonapeptide [27– 30]. Complete characterization of the cloned genes was obtained using an in vivo heat shock experiment. The levels of hsc70 were found to be 8.6-fold higher than hsp70 in control fish and upon heat shock hsc70 was increased 1.7-fold whereas hsp70 increased 6.7-fold. The expression levels of both transcripts returned to near control levels following a 24 h recovery period clearly defining a stress associated role for hsc70 and hsp70. From the data obtained in the first part of this study, we were indeed able to clearly distinguish between constitutive and inducible gene members of the hsp70 family, in sea bream, based on gene length, amino acid sequence at the carboxyl terminus, and expression profiles during acute heat shock. Using an RT-PCR coupled with radioisotope probing, for analysis of hsc70 and hsp70 transcripts, it was found that chronic cold temperature (12 C) acclimation of sea bream resulted in lowered hsc70 and increased hsp70 expression in comparison to fish maintained at a warm temperature of 25 C. The time associated with cold temperature exposure of fish appears to be a key factor that should be given careful consideration as hepatic hsc70 expression did not change during an acute cold temperature shock in carp [25] and sea bream [16] whereas the results obtained in the present study demonstrated changes in hsc70 expression after one month of cold temperature acclimation. The decreased hsc70 expression during cold temperature acclimation could be indicative of a condition of minimal or lowered stress as a similar profile has been reported for sea bream maintained in isoosmotic water, an environment that results in lowered stress as defined by lowered hsc70 and an upregulated somatotropic (growth hormone—IGF1) axis [18]. Indeed, the administration of recombinant growth hormone to silver sea bream has also been previously shown to cause lowered hepatic hsc70 expression [16] implying a regulatory link between environmental condition, growth hormone, and hepatic hsc70 expression. The upregulation of hepatic hsp70 during cold temperature acclimation could have occurred either as a consequence of stress-related protein damage as it has been widely reported that abnormal proteins serve as triggers for hsp70 induction [8] or may be indicative of enhanced cytoprotection as increased hsp70 has also
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been shown to protect cells against apoptosis [31]. Given that the classic stress hormone cortisol remained unchanged whereas the anabolic hormones IGF-I, T3, and T4 were increased during cold temperature acclimation, it is unlikely that the increased hsp70 occurred as a consequence of stress mediated protein damage and as such may be viewed as enhanced cytoprotection. Some causal evidence for a cytoprotective role of increased hsp70 in sea bream has been provided, as growth hormone treatment of whole blood caused an upregulation of hsp70 in parallel with protection against apoptosis [32]. Induction for hsp70 requires the binding of a trimeric heat shock factor 1 (HSF 1) to a heat shock promoter [9] and recent studies, using fish and mammalian models, have provided conclusive evidence for a regulatory link between transcription of hsp70 and transcription of hsf1 [33–35]. The expression of hsf1 in hepatic tissue of sea bream, acclimated to different temperatures, was studied using an RT-PCR approach utilizing specific primers from the DNA binding domain and it was found that the hsf1 transcript profile generally paralleled that of hsp70 transcript with a 2.1-fold increase in sea bream acclimated to cold temperature. The data obtained in the present study suggest that cold-induced upregulation of hsp70 is mediated via an elevated hsf1 transcription. Hormonal status has been demonstrated to be influenced by temperature in fish [13,14,36,37] and in the present study hepatic IGF-1 was measured using RTPCR and it was found that levels of transcript were highest in sea bream acclimated to cold temperature. This finding is contrary to reports from rainbow trout [38] and chinook salmon [39] where increased hepatic IGF-1 transcript correlated with increased water temperature. However, IGF-1 transcript abundance in rainbow trout muscle was increased as water temperature decreased [38]. From several studies on sea bream species it has been shown that increased hepatic IGF-1 is correlated with conditions of minimal stress and enhanced growth [18,40,41] and therefore the increased hepatic IGF-1, at cold temperature, is also indicative of similar physiological conditions. Thyroid hormones have been implicated to play a key role in promoting growth of fish [42] and during stress the circulating levels of these hormones are known to decline [43]. Further support for the conjecture of cold temperature-induced growth, in sea bream, could be seen from measurements of serum thyroid hormones which were both found to be significantly increased during cold temperature acclimation and in rainbow trout low temperature influenced thyroidal hormone status as the highest levels of plasma T3 were found in fish kept at 8 C in comparison to those maintained at 12 C and 16 C [44]. The hormonal changes would probably act in concert to at least partially compensate for the otherwise lowered metabolic rates of ectothermic fish species at low temperatures.
In summary the data presented in this study demonstrate how separate gene members of the hsp70 family, in silver sea bream, are differentially expressed during warm or cold water acclimation. The lowered hsc70 and elevated hsp70 found during cold temperature acclimation could be indicative of a condition of both reduced stress and enhanced cytoprotection, respectively. Interestingly, cold temperature acclimation resulted in higher amounts of hormones involved in anabolic growth and as such a cold water environment may prove to be beneficial for sea bream culture in the future.
Acknowledgments This research was supported by Earmarked Grants for Research CUHK4168/99M and CUHK4264/02M (Research Grants Council, Hong Kong) awarded to N.Y.S.W.
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Cloning and characterization of the hsp70 multigene family from silver sea bream: Modulated gene expression between warm and cold temperature acclimation Eddie E. Deane, Norman Y.S. Woo * Department of Biology, The Chinese University of Hong Kong, Shatin, NT, Hong Kong SAR, China Received 7 March 2005 Available online 17 March 2005
Abstract The genes encoding heat shock cognate 70 (hsc70) and inducible heat shock protein 70 (hsp70) were cloned and characterized from silver sea bream liver. Upon acute heat shock (+7 C), the transcript abundance of hsc70 was increased 1.7-fold whereas the transcript abundance of hsp70 increased 6.7-fold. The chronic acclimation of sea bream to cold temperature (12 C) resulted in a downregulation of hsc70 and an upregulation of hsp70 in comparison to levels in sea bream kept at a warmer temperature (25 C). The expression of heat shock transcription factor I was also increased during cold temperature acclimation. Increased amounts of hepatic insulin-like growth factor 1 transcript, serum thyroxine (T4), and triiodothyronine (T3) were also found during cold temperature acclimation whereas serum cortisol remained unchanged. The results from this study demonstrate how temperature acclimation, in fish, can affect the regulation of the hsp70 multigene family and hormonal factors that are associated with anabolism. 2005 Elsevier Inc. All rights reserved. Keywords: Fish; Temperature; hsc70; hsp70; hsf1; IGF-1; Hormones
Disruption of normal cellular processes can cause the rapid and increased synthesis of a group of proteins belonging to the heat shock protein (HSP) families. Members of these different HSP families are grouped according to molecular size and perform varying and different roles in the cell. The HSP90 family is involved in steroid receptor formation and protein folding [1], the HSP70 family is necessary for translocation and protein folding [2], and HSP60 is involved in protein stability and folding [3,4]. These HSP families are important for immune function [5] and have been demonstrated to be upregulated in fish during stress [6]. Of all the HSP families, HSP70 has been most widely studied as a biomarker of stress [7] and the major inducing factor for HSP70 upregulation is the occurrence of damaged cellular pro-
*
Corresponding author. Fax: +852 26035646. E-mail address: [email protected] (N.Y.S. Woo).
0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.03.039
tein [8]. The HSP70 family is coded for by two different genes, a cognate or constitutive type (hsc70) and an inducible type (hsp70), and both of these genes encode proteins that play key roles in the cell as molecular chaperones [2]. In cells, hsc70 remains unchanged or slightly upregulated upon exposure to stress whereas hsp70 is highly induced, from low basal levels, with transcriptional control mediated via heat shock factor 1 (HSF1) that binds in a trimeric form to a hsp70 gene promoter [9]. As fish are aquatic ectotherms, they have to contend with continuous fluctuations in water temperature and the well-documented response is to modulate profiles of metabolic enzymes [10–12] and alterations in endocrine control are also known to occur [13,14]. Tissue HSP70 could be regulated according to external water temperature, but it has yet to be reported on how the gene expression of both hsc70 and hsp70 as well as hsf1 is adjusted in fish tissues in response to warm and cold water temperature acclimation. In the present study, we report on the
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cloning and characterization of both hsc70 and hsp70 from silver sea bream (Sparus sarba) liver. The gene expression profiles for hsc70, hsp70, and hsf1 from sea bream acclimated between cold water (12 C) and warm water (25 C) are presented. To correlate the heat shock protein response with aspects of growth physiology, gene expression studies for the mitogenic hormone insulin-like growth factor 1 (IGF-1) and serum levels of cortisol, thyroxine, and triiodothyronine are reported. To our knowledge this is the first study to clearly define molecular regulation of hsp70, hsc70, and hsf1 genes in fish acclimated to different water temperatures and demonstrates how the hsp70 multigene family is differentially expressed in fish kept at different temperatures.
lated and subjected to further rounds of screening. After three rounds of screening, 10 putative clones for each gene were selected and converted to plasmid by in vivo excision using Escherichia coli strain BM25.8 (Clontech). Plasmids were cycle sequenced using an ABI PRISM dye terminator kit and reaction products were analyzed on an ABI 310 Genetic Analyzer (Perkin Elmer, USA). RT-PCR analysis of hsc70, hsp70, hsf1, and IGF-1. First strand cDNA was synthesized from 1 lg of total RNA, from each sample, using a Powerscript reverse transcriptase kit (Clontech). First strand cDNA synthesis was allowed to proceed at 42 C for 1 h after which time the reaction was incubated at 70 C for 15 min. For PCR amplification of genes, from first strand cDNA template, oligonucleotide primers were designed from the nucleotide sequences of hsc70 and hsp70 cloned genes, and primers for hsf1 and IGF-1 have previously been used for studies on sea bream [18]. As a normalization control for each RT-PCR, primers specific for 18S rRNA were used [19]. All primers were synthesized by Genset (Singapore) and had the following sequences: 5 0 -ATCAGTGATGACGACAA-3 0 5 0 -TGACCCCCCCCCAGGGGC-3 0 hsp70: 5 0 -ATCAGTGAGGAGGACAAA-3 0 5 0 -CTGGGAGCCGCTTCCTGC-3 0 hsf1: 5 0 -CCCCAGTGGAACCAGCTTCCATG-3 0 5 0 -GGATGTTGGAATTCCGTGTCATC-3 0 IGF-1: 5 0 -AGTGCGATGTGCTGTATC-3 0 5 0 -CAGCTCACAGCTTTGGAA-3 0 18S: 5 0 -GCCAAGTAGCATATGCTTGTCTC-3 0 5 0 -AGACTTGCCTCCAATGGATCC-3 0
hsc70:
Materials and methods Experimental fish and holding conditions. Adult silver sea bream (Sparus sarba) weighing between 200 and 300 g were purchased from a local fish farm and transferred to 1000 L seawater tanks. The water in the tanks was at a temperature of 24–25 C, fully aerated, and fish were fed ad libitum once daily with a formulated diet [15]. Fish were allowed to acclimate to experimental conditions for three weeks prior to temperature exposure. Acute heat shock. Fish were divided into three groups (n = 5) and acclimated to opaque laboratory aquaria for two days prior to heat shock experiments. The seawater in the aquaria was changed daily and one group served as a control with water temperature maintained at 25 C. A second group of fish were subjected to a temperature increase of approximately 0.12 C/min (using an immersion heater) until 32 C was attained. Fish were maintained at this temperature for two hours, killed by spinal transection, and livers were removed. A third group of fish were also subjected to a stress of 32 C, for two hours, after which time the immersion heater was removed and the fish were allowed to undergo a recovery period of 24 h at 25 C prior to liver removal. Total RNA was extracted from the liver tissues using a Qiagen RNeasy mini kit (Qiagen), treated with DNase 1 (Gibco-BRL), quantified spectrophotometrically, and stored at 70 C. Temperature acclimation. Two groups of fish (n = 7 per group) were used for temperature acclimation studies. One group was maintained at 25 C and the second group of was kept at 12 C for one month. The water temperature in both of the tanks was maintained by using a system of water coolers and heaters. The water for each group was aerated and fish were fed ad libitum once daily with a formulated diet. After one month, blood was withdrawn from the caudal vessels, allowed to clot at room temperature for 1 h, and then centrifuged at 10,000g for 10 min to obtain serum. The serum was removed, aliqoutted, and stored at 70 C. After blood withdrawal, fish were killed by spinal transection, and livers were removed and extracted for total RNA as previously described. Cloning and characterization of hsc70 and hsp70 genes. A silver sea bream liver cDNA library was constructed using a SMART cDNA library construction kit (Clontech, USA) and 106 plaques of the library were transferred to Hybond NX plaque lifts (Amersham–Pharmacia Biotech, UK). Plaque lifts were prehybridized for 4 h at 60 C in Rapid-Hyb buffer (Amersham) and then hybridized at 65 C for 16 h with a [32P]dCTP-labeled putative hsc70 clone from sea bream [16] or a putative hsp70 DNA fragment isolated using specific PCR primers designed from the zebrafish hsp70 gene [17]. After hybridization, plaque lifts were washed twice in 2· SSC (1.8%w/v sodium chloride; 0.9%w/v trisodium citrate)/0.1% SDS for 30 min at 65 C and then once in 0.1% SSC/0.1% SDS for 30 min at 65 C. Membranes were exposed to X-ray film (Kodak, USA) and positive plaques were iso-
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(sense) (antisense) (sense) (antisense) (sense) (antisense) (sense) (antisense) (sense) (antisense)
PCRs (50 ll) containing 2 ll of first strand cDNA, 0.2 ll Taq DNA polymerase (Promega, USA; 5 U/ll), 5 ll MgCl2 (25 mM), 5 ll reaction buffer, 0.5 ll dNTP mix (10 mM), and 1 ll of each primer (50 lM) were used. PCR amplification was performed using a PTC-100 thermal cycler (MJ Research, USA) with cycle parameters of 94 C for 1 min, 55 C for 1 min, 72 C for 1 min, and a final extension of 72 C for 4 min. To ensure amplification was at the midpoint of the linear phase of amplification, for each gene, preliminary RT-PCRs were performed. A single PCR product, of expected size, was obtained for each gene of interest and these were subcloned into pCRscript plasmid vector (Stratagene, USA), followed by cycle sequencing to confirm identity. No PCR products were detected from negative controls (reactions without reverse transcriptase added). To confirm the specificity of each RT-PCR and to establish stringent hybridization conditions for subsequent analysis, an aliquot (10 ll) was taken from a number of representative samples and transferred to Hybond-N+ membrane (Amersham). Purified cDNA fragments of each gene were radiolabeled using a Rediprime random labeling kit (Amersham) and used as probes for membrane hybridization. Blots were hybridized at 55 C for 16 h, then washed twice with a 2· SSC/0.1% SDS solution for 30 min, once in 0.1· SSC/0.1% SDS at 65 C for 15 min, air-dried for 15 min, and then autoradiographed at 80 C. From preliminary hybridizations, it was established that probes were specific for corresponding amplified fragments from RT-PCRs and for semi-quantification of transcripts, samples were analyzed together in a single hybridization using DNA dot blots which were prepared using a Bio-Dot microfiltration manifold (Bio-Rad). To test for the linearity of detection during subsequent scanning procedures, PCR amplification products were diluted 5-, 10-, 50-, and 100-fold, prepared and blotted according to instructions supplied with Hybond-N+ membrane (Amersham). Membranes were hybridized and washed as described above and exposed to storage phosphor screens (Molecular Dynamics, USA) for 3 h at room temperature after which time the screens were scanned using the Storm PhosphorImaging system with ImageQuant software (Molecular Dynamics) for quantification of amplified fragment. The abundance of each specific gene fragment was normalized to the corresponding 18S abundance in all samples.
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Serum hormone analysis. Serum cortisol was assayed using a competitive ELISA kit (IBL, Hamburg) previously validated for use with sea bream samples [20]. Serum triiodothyronine (T3) and thyroxine (T4) were assayed using competitive ELISA kits (Biomerica, USA) that have been previously validated for use with sea bream samples [20]. Statistical analysis. Normalized transcript abundance, for each gene studied, were subjected to either a one way ANOVA to test for significance followed by a Student–Newman–Keuls test (Jandel Scientific) or a StudentÕs t test to delineate significance between groups. Significant differences were accepted if p < 0.05.
Results Isolation and characterization of hsc70 and hsp70 genes Using specific gene fragments, as radiolabeled probes against a liver cDNA library, 10 putative clones for hsc70 and hsp70 were isolated. The sea bream hsc70 open reading frame was 1950 bp in length coding for a protein of 649 amino acids (Fig. 1) whereas the sea
Fig. 1. Nucleotide and deduced amino acid sequence of silver sea bream heat shock cognate 70 (hsc70). The nucleotides are numbered on the left and the amino acids are numbered on the right. An asterisk above the last three nucleotides indicates a stop codon. The sequence encoding sea bream hsc70 has been deposited with the GenBank under Accession No. AY436786.
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bream hsp70 open reading frame was 1920 bp in length coding for a protein of 639 amino acids (Fig. 2). The amino acid homology between sea bream hsc70 and hsp70 was 85% which was to be expected since both genes belong to the same multigene family. After sequence comparisons using the Basic Local Alignment Search Tool Program [21], it was found that sea bream
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hsc70 shared 80–91% amino acid homology with many other animal hsc70 genes and similarly sea bream hsp70 shared a 77–86% amino acid homology with many other animal hsp70 genes. The sequences encoding sea bream hsc70 and hsp70 have been deposited with the GenBank under Accession Nos. AY436786 and AY436787, respectively.
Fig. 2. Nucleotide and deduced amino acid sequence of silver sea bream inducible heat shock protein 70 (hsp70). The nucleotides are numbered on the left and the amino acids are numbered on the right. An asterisk above the last three nucleotides indicates a stop codon. The sequence encoding sea bream hsp70 has been deposited with the GenBank under Accession No. AY436787.
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Expression analysis of hsc70 and hsp70 during acute heat shock The effects of an acute heat shock on hepatic hsc70 and hsp70 expression were studied, using an RT-PCR assay. The transcript abundance of hsc70 increased 1.7-fold following heat shock and then returned to similar levels as that found in non-heat-shocked fish, after a 24 h recovery period. The transcript abundance of hepatic hsp70 increased 6.7-fold following acute heat shock and also returned to similar levels as that found in non-heat-shocked fish, after a 24 h recovery period. These results are presented in Fig. 3.
(Fig. 4). Sea bream at 12 C also had 2.1-fold higher amounts of hsf1 transcript in comparison to fish acclimated to 25 C (Fig. 4). The levels of IGF-1 transcript were also found to be 2-fold higher in 12 C acclimated fish (Fig. 5).
Expression analysis of hsc70, hsp70, hsf1, and IGF-1 during warm and cold temperature acclimation Using an RT-PCR coupled with radioisotope probing approach, the transcript amounts of hsc70, hsp70, hsf1, and IGF-1 genes could be studied in liver tissue from sea bream acclimated to warm (25 C) or cold (12 C). The transcript abundance of hsc70 was found to be 2.3-fold higher in fish kept at 25 C in comparison to 12 C acclimated fish whereas the transcript abundance of hsp70 displayed an opposite trend with 2-fold higher levels at 12 C in comparison to fish at 25 C
Fig. 4. Expression of hepatic hsc70, hsp70, and hsf1 in sea bream acclimated to 12 and 25 C. RT-PCR was used to amplify transcripts and phosphorimaging analysis was used for transcript quantification. Data are presented as mean normalized transcript abundance (n = 7) ± SEM and an asterisk above the bar denotes a significantly different mean value (p < 0.05).
Fig. 3. Expression of hepatic hsc70 and hsp70 during acute heat shock of sea bream in vivo. Fish were kept at control (25 C) or subjected to an acute heat shock (HS) for 2 h at 32 C or subjected to a heat shock and then allowed to recover, at control temperature for 24 h (HS/R). Transcripts for all samples were quantified using phosphorimaging and the amount of hsc70 and hsp70 transcripts was normalized to the corresponding 18S transcript abundance of each sample and expressed in arbitrary units as mRNA abundance. All values are expressed as means ± SEM (n = 5) and different letters above a bar mean indicate values which are significantly different from each other (p < 0.05).
Fig. 5. Expression of hepatic IGF1 in sea bream acclimated to 12 and 25 C. RT-PCR was used to amplify transcripts and phosphorimaging analysis was used for transcript quantification. Data are presented as mean normalized transcript abundance (n = 7) ± SEM and an asterisk above the bar denotes a significantly different mean value (p < 0.05).
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Fig. 6. Measurements of serum cortisol (A), serum T4 (B), and T3 (C) in silver sea bream acclimated to 12 and 25 C. Serum cortisol is expressed as nanogram per milliliter and T4 and T3 are expressed as lg/100 ml. Data are presented as mean values (n = 7) ± SEM and an asterisk above a bar denotes a significantly higher mean value (p < 0.05).
Serum cortisol, T4, and T3 analysis The serum levels of cortisol remained unchanged between sea bream at 12 C (10.6 ± 1.2 ng/ml) and 25 C (11.0 ± 1.5 ng/ml). The amounts of serum T4 were 1.9fold higher in fish acclimated to 12 C (8.7 ± 1.4 lg/ 100 ml) than 25 C (4.6 ± 1.1 lg/100 ml) and similarly the amount of serum T3 was 1.8-fold higher in fish at 12 C (4.0 ± 0.31 lg/100 ml) than 25 C (2.2 ± 0.3 lg/ 100 ml). These data are presented in Fig. 6.
Discussion Presently only a relatively small number of genes from the hsp70 family in fish have been cloned and characterized (see [22] for review), and a clear delineation between constitutive and inducible members of the hsp70 family, in teleosts acclimated between warm and cold temperature, remains to be elucidated. To address this present void, both constitutive (hsc70) and inducible (hsp70) genes from sea bream liver were cloned and
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characterized in this study. The amino acid homology between hsc70 and hsp70 was high but analysis of the heterogeneous carboxyl regions of hsc70 and hsp70 in fish defines certain features that allowed us to differentiate between these two genes. The constitutive hsc70 genes are slightly longer than hsp70 genes and the carboxyl region of these genes is highly heterogeneous as hsc70 displays a series of unique repeat GGXP motifs and a signature nonapeptide SGPTIEEVD at the end [23–26]. In fish, inducible hsp70 genes, the GGXP motif occurs once in most cases and the last nine amino acids are not the same as the terminal hsc70 nonapeptide [27– 30]. Complete characterization of the cloned genes was obtained using an in vivo heat shock experiment. The levels of hsc70 were found to be 8.6-fold higher than hsp70 in control fish and upon heat shock hsc70 was increased 1.7-fold whereas hsp70 increased 6.7-fold. The expression levels of both transcripts returned to near control levels following a 24 h recovery period clearly defining a stress associated role for hsc70 and hsp70. From the data obtained in the first part of this study, we were indeed able to clearly distinguish between constitutive and inducible gene members of the hsp70 family, in sea bream, based on gene length, amino acid sequence at the carboxyl terminus, and expression profiles during acute heat shock. Using an RT-PCR coupled with radioisotope probing, for analysis of hsc70 and hsp70 transcripts, it was found that chronic cold temperature (12 C) acclimation of sea bream resulted in lowered hsc70 and increased hsp70 expression in comparison to fish maintained at a warm temperature of 25 C. The time associated with cold temperature exposure of fish appears to be a key factor that should be given careful consideration as hepatic hsc70 expression did not change during an acute cold temperature shock in carp [25] and sea bream [16] whereas the results obtained in the present study demonstrated changes in hsc70 expression after one month of cold temperature acclimation. The decreased hsc70 expression during cold temperature acclimation could be indicative of a condition of minimal or lowered stress as a similar profile has been reported for sea bream maintained in isoosmotic water, an environment that results in lowered stress as defined by lowered hsc70 and an upregulated somatotropic (growth hormone—IGF1) axis [18]. Indeed, the administration of recombinant growth hormone to silver sea bream has also been previously shown to cause lowered hepatic hsc70 expression [16] implying a regulatory link between environmental condition, growth hormone, and hepatic hsc70 expression. The upregulation of hepatic hsp70 during cold temperature acclimation could have occurred either as a consequence of stress-related protein damage as it has been widely reported that abnormal proteins serve as triggers for hsp70 induction [8] or may be indicative of enhanced cytoprotection as increased hsp70 has also
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been shown to protect cells against apoptosis [31]. Given that the classic stress hormone cortisol remained unchanged whereas the anabolic hormones IGF-I, T3, and T4 were increased during cold temperature acclimation, it is unlikely that the increased hsp70 occurred as a consequence of stress mediated protein damage and as such may be viewed as enhanced cytoprotection. Some causal evidence for a cytoprotective role of increased hsp70 in sea bream has been provided, as growth hormone treatment of whole blood caused an upregulation of hsp70 in parallel with protection against apoptosis [32]. Induction for hsp70 requires the binding of a trimeric heat shock factor 1 (HSF 1) to a heat shock promoter [9] and recent studies, using fish and mammalian models, have provided conclusive evidence for a regulatory link between transcription of hsp70 and transcription of hsf1 [33–35]. The expression of hsf1 in hepatic tissue of sea bream, acclimated to different temperatures, was studied using an RT-PCR approach utilizing specific primers from the DNA binding domain and it was found that the hsf1 transcript profile generally paralleled that of hsp70 transcript with a 2.1-fold increase in sea bream acclimated to cold temperature. The data obtained in the present study suggest that cold-induced upregulation of hsp70 is mediated via an elevated hsf1 transcription. Hormonal status has been demonstrated to be influenced by temperature in fish [13,14,36,37] and in the present study hepatic IGF-1 was measured using RTPCR and it was found that levels of transcript were highest in sea bream acclimated to cold temperature. This finding is contrary to reports from rainbow trout [38] and chinook salmon [39] where increased hepatic IGF-1 transcript correlated with increased water temperature. However, IGF-1 transcript abundance in rainbow trout muscle was increased as water temperature decreased [38]. From several studies on sea bream species it has been shown that increased hepatic IGF-1 is correlated with conditions of minimal stress and enhanced growth [18,40,41] and therefore the increased hepatic IGF-1, at cold temperature, is also indicative of similar physiological conditions. Thyroid hormones have been implicated to play a key role in promoting growth of fish [42] and during stress the circulating levels of these hormones are known to decline [43]. Further support for the conjecture of cold temperature-induced growth, in sea bream, could be seen from measurements of serum thyroid hormones which were both found to be significantly increased during cold temperature acclimation and in rainbow trout low temperature influenced thyroidal hormone status as the highest levels of plasma T3 were found in fish kept at 8 C in comparison to those maintained at 12 C and 16 C [44]. The hormonal changes would probably act in concert to at least partially compensate for the otherwise lowered metabolic rates of ectothermic fish species at low temperatures.
In summary the data presented in this study demonstrate how separate gene members of the hsp70 family, in silver sea bream, are differentially expressed during warm or cold water acclimation. The lowered hsc70 and elevated hsp70 found during cold temperature acclimation could be indicative of a condition of both reduced stress and enhanced cytoprotection, respectively. Interestingly, cold temperature acclimation resulted in higher amounts of hormones involved in anabolic growth and as such a cold water environment may prove to be beneficial for sea bream culture in the future.
Acknowledgments This research was supported by Earmarked Grants for Research CUHK4168/99M and CUHK4264/02M (Research Grants Council, Hong Kong) awarded to N.Y.S.W.
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