Comparative Biochemistry and Physiology Part A 134 (2003) 839–846 Stressor-dependent regulation of the heat shock response in zebrafish, Danio rerio a,1 Susanna Airaksinena,*, Christina M.I. Rabergh , Anna Lahtia, Annukka Kaatrasaloa, ˚ Lea Sistonenb,c, Mikko Nikinmaaa b a Department of Biology, Laboratory of Animal Physiology, University of Turku, FIN-20014 Turku, Finland ˚ Akademi University, P.O. Box 123, FIN-20521 Turku, Finland Turku Centre for Biotechnology, University of Turku, Abo c ˚ Akademi University, Turku, Finland Department of Biology, Abo Received 18 November 2002; received in revised form 21 January 2003; accepted 22 January 2003 Abstract Heat shock transcription factors (HSFs) regulate expression of heat shock proteins (Hsps). We have previously shown that in zebrafish a unique isoform, zHSF1b, disappears concomitant with heat shock-induced Hsp70 expression. To characterize the role of zHSF1a and zHSF1b isoforms in the regulation of the stress response in vivo, we have carried out cadmium (10–100 mM) and copper (10–30 mM) exposures in order to specify whether the disappearance of HSF1b is specific for heat stress. After 4-h metal exposures we analyzed the expression of hsp70, zHSF1a, zHSF1b and metallothionein (MT) by reverse transcriptase polymerase chain reaction in zebrafish liver, gonads and gills. Although cadmium is a known inducer of Hsps, it did not affect hsp70 expression significantly in the studied tissues. Induction of hsp70 was observed upon copper exposure in liver and gonads, but not in gills. Neither metal affected the zHSF1ay b ratio. Both cadmium and copper exposure caused upregulation of MT, regulator of metal homeostasis and detoxification, confirming that the tissues were subjected to metal loads. Thus, hsp70 appears to be more weakly induced upon metal exposure than in response to heat shock and HSF1 isoforms may participate in stressor-specific regulation of hsp70. 䊚 2003 Elsevier Science Inc. All rights reserved. Keywords: Cadmium; Copper; Danio rerio; Heat shock; Heat shock factor; Heat shock protein; Metallothionein; Zebrafish 1. Introduction Diverse environmental signals trigger heat shock transcription factor (HSF)-mediated activation of heat shock (HS) genes encoding heat shock proteins (Hsps). These stress proteins play a central role in cell protection and repair upon stress as *Corresponding author. Finnish Game and Fisheries ¨ Research Institute, Turku Game and Fisheries Research, Itainen ¨ Pitkakatu 3, FIN-20520 Turku, Finland. Tel.: q358-205751688; fax: q358-205-751689. E-mail address: susanna.airaksinen@rktl.fi (S. Airaksinen). 1 Present address: Leiras Oy, Pansiontie 47, FIN-20101 Turku, Finland. well as under certain non-stressful conditions (for reviews, see Morimoto, 1998; Feder and Hofmann, 1999; Basu et al., 2002). Among the reported inducers of Hsps are heavy metals (Heikkila et al., 1982; Misra et al., 1989; Fischbach et al., 1993; Wagner et al., 1999), which are known to induce also another set of proteins, metallothioneins (MTs) (Durnam and Palmiter, 1981). MTs are involved in metal homeostasis and detoxification (Palmiter, 1998), and they are often classified as a specific family of stress proteins, since they are induced not only by metals, but also by cytokines (Karin et al., 1985; De et al., 1990), mitogens (Imbra and Karin, 1987), and glucocorticoids 1095-6433/03/$ - see front matter 䊚 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S1095-6433(03)00017-5 840 S. Airaksinen et al. / Comparative Biochemistry and Physiology Part A 134 (2003) 839–846 (Karin and Herschman, 1979). However, MTs are not structurally related to the classical members of Hsp families. Instead, their peculiar cysteine-rich structure is specialized for high affinity metal binding and to serve as a protein thiol source within a cell (for review see, Klaassen et al., 1999). The promoter region of HS genes contains a conserved heat shock element (HSE), which is responsible for transcriptional regulation via HSFbinding. It has been shown that also in fish the HSE is occupied by HSF upon hsp70 induction (Airaksinen et al., 1998). Among the several members of the HSF family in vertebrates discovered to date, HSF1 is the prototype of the stressresponsive HSF (for review, see Pirkkala et al., 2001) and the functional homologue of a single HSF in yeast (Wiederrecht et al., 1988; Sorger and Pelham, 1988) and fruit fly (Clos et al., 1990). We have previously cloned HSF1 from zebrafish and shown that two isoforms HSF1a and HSF1b exist (Rabergh ˚ et al., 2000). Recently, an additional isoform, HSF1c, has been reported in zebrafish (Wang et al., 2001). Interestingly, the zHSF1b isoform disappears following HS with concomitant induction of Hsp70, the major heat-inducible Hsp (Rabergh ˚ et al., 2000). This observation raised the question of the role of the zebrafish HSF1 isoforms in the regulation of the stress response upon exposure to stressors other than heat. Since both Hsp and MT expression can be triggered by common stressors, such as oxidative stress or heavy metals (Andrews, 2000; Gosslau et al., 2001), the present study was designed to characterize the role of zHSF1 isoforms upon metal stress in zebrafish in vivo. Cadmium and copper exposures were carried out in order to specify whether the disappearance of HSF1b is stressordependent. The results suggest that the role of HSF1 isoforms in the regulation of metal inducible hsp70 expression is distinct from that observed upon heat stress, where HSF1ayHSF1b ratio is dramatically changed upon hsp70 induction ˚ (Rabergh et al., 2000). Furthermore, high sublethal concentrations of cadmium and copper cause only a minor increase in the hsp70 mRNA and protein levels in zebrafish tissues although the MT expression is readily induced. 2. Materials and methods 2.1. Animals and experimental design Adult zebrafish (Danio rerio) were obtained from a local aquarium store and allowed to acclimatize to laboratory conditions for a minimum of 2 weeks prior to the experiments. Fish were maintained at 28 8C in dechlorinated carbon filtered Turku tap water (wNaqxf8.8 mgyl, wClyxf24.8 wCd2qxf0.001 mgyl, mgyl, wCu2qxf0.022 mgyl, wCaCO3xf0.69 mmolyl, pH 7.0) in 12 h:12 h light–dark cycle and fed daily (TetraMin, TetraWerke, Germany). Before the metal exposure, fish were transferred to aerated 500ml glass containers with a water temperature of 28 8C, and allowed to recover for an hour. Thereafter, the required volume of water was substituted with 10 mM metal solution (CdCl2 and CuSO4 in double distilled water), so that the desired concentrations were obtained. Double distilled water was used for the controls in order to cause a similar physical disturbance to both experimental and control groups. According to pilot experiments the exposure time and the concentrations showing the maximal hsp70 induction (without recovery period) were chosen to be analyzed in detail, i.e., 4-h exposure with 50 mM CdCl2 (5.6 mgyl) and 4-h exposure with 20–30 mM CuSO4 (1.3–1.9 mgyl). Each treatment was repeated 2–4 independent times. Immediately after the 4-h exposure fish were decapitated, and the gills, female gonads and liver were dissected on ice. 2.2. Reverse transcriptase polymerase chain reaction Liver and gill samples were pooled from three to five fish and gonad samples from one to two fish. Tissues were homogenized on ice immediately after dissection (ULTRA-TURRAX, Ika Labortechnik, Germany) in RNAzolB solution (Tel-Test, Inc., Friendswood, TX). Total RNA was isolated using the RNAzolB method according to the manufacturer’s instructions. Synthesis of cDNA was performed with 5 mg of RNA using general oligo(dT)15-primer (Promega, Madison, WI), together with avian myeloblastosis virus reverse transcriptase (Finnzymes, Espoo, Finland). Subsequent amplification reactions with gene-specific primers of zebrafish HSF1a, HSF1b, hsp70, and hsc70 were performed as described earlier (Rabergh et al., 2000). The amplified products ˚ obtained were 605, 700, 457, and 412 bp, respectively. MT was amplified using primers designed based on the zebrafish MT sequence available in GenBank (accession no. NM_131075; forward, 59- S. Airaksinen et al. / Comparative Biochemistry and Physiology Part A 134 (2003) 839–846 atggatccttgcgaatg-39; reverse, 59-tcactgacaacagctgg39). The optimum annealing temperature for the MT-primers was determined to be 47 8C with the gradient program (HYBAID thermal cycler) and the product size was 183 bp. In order to confirm that the obtained sequence was MT, the product was sequenced (ABI Prism 377, Perkin Elmer, UK). The PCR-products were run on 1.4% TBEagarose gels, whereafter the gels were photographed under UV-light. The unsaturated bands were quantified using Chemi-imager with AlphaEase娃 software (Alpha Innotech Corporation, San Leandro, CA) with respect to Hsc70, which is an evenly and ubiquitously expressed member of hsp70 family. Generally, 3–4 independent samples were analyzed, and the statistical significance of the differences tested with t-test. However, due to the lack of mature female fish among 50 mM cadmium-treated fish Hsp70 and MT transcript levels were obtained only from one and two individual gonad samples, respectively. 2.3. Western blot analysis Whole cell extracts were prepared from gills, gonads and livers by a modification of the method described by Mosser et al. (1988). Briefly, dissected tissues, which were kept on ice throughout the procedure, were homogenized (ULTRATURRAX) and sonicated with Branson microtip sonicator (G. Heinemann, Ultraschalltechnik, Germany) in buffer containing 25% glycerol (vyv), 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 20 mM HEPES, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiotreitol. Thereafter, samples were centrifuged for 30 min (4 8C, 14 000=g) and the supernatants were collected and stored at y70 8C. The protein concentration of the whole cell extracts was determined using the Bio-Rad protein assay according to the manufacturer’s instructions (Bio-Rad, California, USA). Equal amounts of protein (20 mg) were loaded on a discontinuous SDS-polyacrylamide gel (8%), separated and transferred to nitrocellulose membrane (Schleicher & Schuell) using a semidry transfer apparatus (Bio-Rad). The membranes were blocked as described earlier (Rabergh et al., ˚ 2000). Hsp70 protein was detected using a monoclonal mouse anti-HSP70 antibody (clone 3a3, Affinity Bioreagents, Golden, CO) diluted 1:10 000. b-Actin was detected using a mouse anti-b-actin antibody (clone AC-15, Sigma- 841 Aldrich, Inc.) diluted 1:2000. In order to perform enhanced chemiluminescence (Amersham Pharmacia Biotech UK Limited), the horseradish peroxidase-conjugated rabbit anti-mouse immunoglobulin (Amersham) was used as a secondary antibody. The signal was captured on X-ray film and results were photographed and analyzed with Chemi-imager (Alpha Innotech Corp.). Three separate experiments were carried out with cadmium and two with copper. 3. Results 3.1. The ratio of zHSF1ayzHSF1b isoforms remains unaltered following a short term cadmium exposure Cadmium is a potent inducer of Hsp70 as reported in numerous studies on mammalian as well as on fish cell cultures (Levinson et al., 1980; Heikkila et al., 1982; Misra et al., 1989; Fischbach et al., 1993; Steiner et al., 1998). We exposed zebrafish to cadmium for 4 h, after which the expression of HSF1, hsp70, and hsc70 (constitutive form of Hsp70) was analyzed by RT-PCR. In liver and gills cadmium had no significant effect on the hsp70 expression as determined based on three and four independent experiments, respectively (Fig. 1a). However, when change occurred it was upregulation of hsp70. Also in the only gonad sample obtained at a cadmium concentration of 50 mM increase in the hsp70 expression was observed and the increase was evident already at 20 mM cadmium concentration (data not shown). The absence of significant induction was further confirmed with northern blot analysis (data not shown). Western blotting was used to analyze Hsp70 protein levels (Fig. 1b). In agreement with the RTPCR data, there was no detectable increase in Hsp70 in the studied tissues when normalized against b-actin (ns3). Again, however, in one or two cases slight induction was observed in liver and gonads, respectively. As a comparison, an increased Hsp70 level in zebrafish liver was shown after 1-h HS (9 8C above the control temperature). As previously reported, heat-inducible isoform of Hsp70 is detected in gills and liver, and to a lesser extent in gonads upon HS (Rabergh et al., 2000), ˚ indicating tissue-specificity of the temperatureinduced responses. 842 S. Airaksinen et al. / Comparative Biochemistry and Physiology Part A 134 (2003) 839–846 Fig. 1. (a) Expression of hsp70, two zHSF1 isoforms a and b, and hsc70 following cadmium exposure (4 h) presented by RT-PCR. Gene-specific primers were used to study different tissues, i.e., liver, gonads and gills. The presented samples were pooled from five fish, except 0 and 50 mM gonad samples, which were obtained from three and one female, respectively. Hsp70yHsc70 and HSF1ayb ratios are shown below the panels. (b) Western blot analysis of zebrafish exposed to cadmium. Hsp70 (above) and b-actin (below) were analyzed in liver, gonads and gills. The samples were pooled from four fish, except the gonad samples, which were pooled from two fish. Heat-shocked (1 h, 37 8C) zebrafish liver sample is designated as HS. (c) Expression of MT and hsc70 presented as described above. The presented samples were pooled from five fish, except 0 and 50 mM gonad samples, which were obtained from two and one female, respectively. MTyHsc70 ratios are shown below the panels. (d) Dependency of MT expression on cadmium concentration in liver (pooled from five fish) presented as described above. Cadmium concentrations are indicated above each panel and MTyHsc70 ratios are shown as in (c). Since Hsp70 was poorly induced by cadmium, which is generally considered as a potent inducer of the HS response, we investigated whether the concentrations of metal used in the exposures were adequate to cause an upregulation of MT, a common indicator for metal exposure (Samson and Gedamu, 1998). Cadmium, which caused only a minor (liver and gonads) or hardly detectable (gills) induction of hsp70, caused a prominent induction of MT in liver (P-0.05, ns4) and gills (P-0.05, ns4) at 50 mM concentration (Fig. 1c). As shown in Fig. 1d, MT levels increased in a dose-dependent manner. In contrast, the expression level of MT in gonads was unaffected (ns2) (Fig. 1c). 3.2. Hsp70 is more efficiently induced than MT by copper We analyzed the expression of HSF1, hsp70, and hsc70 following a 4-h exposure to copper, an essential metal, which is a cofactor of many enzymes and is also known to induce MTs (Durnam and Palmiter, 1981; Nieminen and Lemasters, 1996). The hsp70 expression was moderately increased at copper concentrations of 25 mM in liver (P-0.1, ns4) and gonads (P-0.06, ns3), whereas the expression in gills remained unaffected (ns3)(Fig. 2a). Similar to the cadmium treatments, no change was observed in the ratio of HSF1 isoforms in any of the tissues (Fig. 2a). Western blot analysis suggests comparable Hsp70 increase in liver and gonads following copper exposure (ns2) (Fig. 2b). As a comparison, a markedly elevated Hsp70 protein level in liver induced by HS is shown in Fig. 2b. To avoid further stress for experimental fish due to handling no recovery period after the exposure was allowed. This may limit the degree of induction at the protein level. Next we studied whether the concentrations of copper used in this study were sufficient to induce the expression of MT. In the liver, a slight increase in MT expression (P-0.1, ns4) was observed at S. Airaksinen et al. / Comparative Biochemistry and Physiology Part A 134 (2003) 839–846 843 study upon heat-inducible hsp70 induction (Rabergh et al., 2000). Accordingly, the HSF1b ˚ isoform disappeared gradually at 35–37 8C (upper panel in Fig. 3). The pattern of expression upon metal exposure was, however, distinct from that observed upon heat stress (lower panel in Fig. 3). Neither of the metals affected the HSF1ayb ratio even though the target gene was upregulated by copper (Fig. 2). In gonads, zHSF1a and zHSF1b were equally expressed regardless of the treatment, and in the liver both isoforms were present at control temperature and both in cadmium and copper-treated animals, whereas zHSF1b disappeared during HS. In gills zHSF1a was a dominant isoform under all conditions (Fig. 1a and Fig. 2a). 4. Discussion 4.1. Cadmium and copper as stress inducers Fig. 2. (a) Expression of hsp70, two zHSF1 isoforms a and b, and hsc70 following copper exposure (4 h) presented by RTPCR. Gene-specific primers were used to study different tissues, i.e., liver, gonads and gills. The samples were pooled from five, two and five fish, respectively. Hsp70yHsc70 and HSF1ayb ratios are shown below the panels. (b) Western blot analysis of zebrafish exposed to copper. Hsp70 (above) and bactin (below) were analyzed in liver, gonads and gills. The samples were pooled from four fish, except the gonad samples, which were pooled from one, two and four fish, respectively. Heat shocked (1 h, 37 8C) zebrafish liver sample is designated as HS. (c) Expression of MT and hsc70 presented as described above. The liver sample was pooled from five, the gonad sample from two, and the gill sample from five fish. Copper concentrations are indicated above each panel and MTyHsc70 ratios are shown as in Fig. 1c. concentration of 25 mM suggesting an adequate copper load (Fig. 2c). Indeed, higher concentrations of copper (035 mM) proved to be lethal to the fish, indicating the severity of the stress (data not shown). The tissue-specific response was reflected in the observation that MT induction was reversed in gonads (P-0.05, ns4) and absent in gills (ns3) (Fig. 2c). 3.3. Stressor-specific regulation of the heat shock response by zHSF1 isoforms A dramatic change in the ratio of HSF1 isoforms was observed in zebrafish liver in our previous Surprisingly, well known inducers of HS response proved to be only modest inducers of Hsp70 in adult zebrafish liver, gonad and gill tissue. The observed induction of hsp70 following HS could be mediated by stimulating the activatory effect of zHSF1a andyor eliminating the inhibitory effect of zHSF1b. Neither cadmium nor copper caused a change in the zHSF1ayb ratio. If a change in the HSF isoform ratio is indeed required for full hsp70 induction, the unaltered ratio could be reflected in the diminished Hsp70 induction in response to metal treatments, when compared to Fig. 3. Comparison of zHSF1a and zHSF1b expression after 1 h HS (upper panel) or after 4 h cadmium and copper exposure (lower panels). HS temperatures (33, 35, or 37 8C) and concentrations of cadmium and copper treatments (28 8C) are indicated above the panels. The ratios of HSF1ayHSF1b expression following treatments as obtained by image analysis are shown below the panels. The presented samples were pooled from five fish. 844 S. Airaksinen et al. / Comparative Biochemistry and Physiology Part A 134 (2003) 839–846 HS. The binding of zHSF1 to the intact hsp70 promoter was not analyzed in current study. Wang et al. have, however, reported enhanced binding of in vitro translated zHSF1a and zHSF1c to HSE at elevated temperature (Wang et al., 2001). An explanation for the profound induction of MT concurrent with the minor HS response could be that the metal load was sequestered by MTs (Foulkes and McMullen, 1986). However, it has been shown that at least in zebrafish gills the cadmium-binding capacity, including MT-binding, is exceeded at as low as 15 nM concentration of cadmium, whereupon influx into circulation is drastically increased (Wicklund, 1996). Therefore, current results may also reflect variability of different tissues in adjusting the response to a given stressor. The metal inducible response appeared to follow tissue-specific patterns, as observed before with heat stress (Rabergh et al., 2000). It is ˚ noteworthy that the sublethal metal concentrations used in this study correspond to the concentrations used in cell cultures, where they have been shown to induce the HS response (Heikkila et al., 1982; Misra et al., 1989; Ryan and Hightower, 1994; Croute et al., 2000). Furthermore, the concentrations used were close to the upper limits tolerated by zebrafish in vivo. A recent study by Blechinger et al. (2002) has monitored the cadmium-induced hsp70 expression in early larvae (80 h) of zebrafish. Interestingly, both endogenous hsp70 expression and reporter gene expression under hsp70 promoter showed dose-dependent increase in gills (00.2 mM) and liver (0125 mM), which adds the life-stage specificity to the list of variability creating factors. MT protects the animal most effectively against cadmium toxicity, when compared to other metals such as copper, zinc, iron, lead, mercury, or arsenite (Park et al., 2001). Also, cadmium has been shown to be a better inducer of MT in bovine chondrocytes and mouse tissues compared to copper (Durnam and Palmiter, 1981; Zafarullah et al., 1993). Furthermore, when the metal regulatory element (MRE) driven luciferase activity was measured in zebrafish cell line, ZEM2S, upon a number of metal treatments, the inducibility was lowest with copper and highest with cadmium (Carvan et al., 2000). Observations above correspond to the difference in MT induction observed in this study between cadmium and copper treatment. The tissue distribution of metals was not measured in the present study. However, depending on the cadmium-sensitivity of the fish species liver, kidney and gills are known to accumulate most of the cadmium in the course of time (Norey et al., 2002). 4.2. Is there a signaling network connecting heat and metal stress? A common regulatory element in the promoter of HS genes, HSE, is also found in the superoxide dismutase (SOD1) promoter, where it is occupied upon treatment by two distinct stressors, paraquat, a Oy 2 generating agent, and HS (Yoo et al., 1999). The communication in this case appears to occur at the level of transcription, and may be mediated through the same signaling molecule, i.e., superoxide. This suggests an involvement of redox reactions in the process. Interestingly, many metals, such as copper, are capable of changes in valency thus affecting the cellular redox status. Both Hsp70 and MT promoters have been studied intensively in order to find out whether the regulatory regions of these genes might also have common elements. It appears, however, that this is not the case. Although the MT gene expression is also regulated at the transcriptional level, the MRE recruited by a specific metal transcription factor, MTF-1, upon activation is distinct from HSE (Stuart et al., 1985; Radtke et al., 1993; Olsson et al., 1995; Dalton et al., 1997; Samson et al., 2001; Chen et al., 2002). 4.3. Stress response at cellular vs. organismic level Numerous studies performed on cell cultures as well as general statements about the HS response suggest that the Hsps are prominently induced by heavy metals. Based on our results with cadmium where MTs were induced in vivo in the virtual absence of HS response, this statement may be too simplistic in biologically relevant context. The nature of the stressor and its signaling pathway within each tissue of an organism should therefore be taken into account when estimating the biological significance of a specific type of stress to its target. In conclusion, in the studied tissues of adult zebrafish Hsp70 appears to be weakly, if at all induced upon metal exposures, compared to the marked induction observed in response to heat stress. Since the zHSF1-isoform ratio is markedly changed as a response to elevated temperatures, but remains unaffected by metals, this stressor- S. Airaksinen et al. / Comparative Biochemistry and Physiology Part A 134 (2003) 839–846 specific response of HSF may be an important determinant in the induction of HS response in intact animal tissues when exposed to different stressors. This study highlights the great complexity of the stress response, which becomes apparent only when experiments are conducted at the organismic level instead of isolated cells in culture. Acknowledgments This work was supported by the Academy of Finland, projects 40830, 42186 and 50748. References Airaksinen, S., Rabergh, C.M.I., Sistonen, L., Nikinmaa, M., ˚ 1998. 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