Constitutive heat shock protein 70 (HSC70) expression in rainbow trout hepatocytes: effect of heat shock and heavy metal exposure

Comparative Biochemistry and Physiology Part C 132 (2002) 223–233

Constitutive heat shock protein 70 (HSC70) expression in rainbow trout hepatocytes: effect of heat shock and heavy metal exposure Adrienne N. Boone, Mathilakath M. Vijayan* Department of Biology, University of Waterloo, Waterloo, Ont., Canada N2L 3G1 Received 28 October 2001; received in revised form 11 April 2002; accepted 12 April 2002

Abstract The 70-kDa family of heat shock proteins plays an important role as molecular chaperones in unstressed and stressed cells. The constitutive member of the 70 family (hsc70) is crucial for the chaperoning function of unstressed cells, whereas the inducible form (hsp70) is important for allowing cells to cope with acute stressor insult, especially those affecting the protein machinery. In fish, the role of hsc70 in the cellular stress response process is less clear primarily because of the lack of a fish-specific antibody for hsc70 detection. In this study, we purified hsc70 to homogeneity from trout liver using a three-step purification protocol with differential centrifugation, ATP-agarose affinity chromatography and electroelution. Polyclonal antibodies to trout hsc70 generated in rabbits cross-reacted strongly with both purified trout hsc70 protein and also purified recombinant bovine hsc70. Two-dimensional electrophoresis followed by Western blotting confirmed that the isoelectric point of rainbow trout hsc70 was more acidic than hsp70. Using this antibody, we detected hsc70 content in the liver, heart, gill and skeletal muscle of unstressed rainbow trout. Primary cultures of trout hepatocytes subjected to a heat shock (q15 8C for 1 h) or exposed to either CuSO4 (200 mM for 24 h), CdCl2 (10 mM for 24 h) or NaAsO2 (50 mM for 1 h) resulted in higher hsp70 accumulation over a 24-h period. However, hsc70 content showed no change with either heat shock or heavy metal exposure suggesting that hsc70 is not modulated by sublethal acute stressors in trout hepatocytes. Taken together, we have for the first time generated polyclonal antibodies specific to rainbow trout hsc70 and this antibody will allow for the characterization of the role of hsc70 in the cellular stress response process in fish. 䊚 2002 Elsevier Science Inc. All rights reserved. Keywords: Heat shock protein 70; Liver; Hsc70 antibody; Trout; Fish; Oncorhynchus mykiss; Stress; Copper; Cadmium; Arsenite

1. Introduction Heat shock proteins (hsps) are a family of highly conserved proteins playing an important role in the functioning of unstressed and stressed cells (Parsell and Lindquist, 1993). The hsp70 family, the most widely studied of the hsps, is constitutively expressed (hsc70) in unstressed cells and is also induced in response to stressors (hsp70), especially those affecting the protein *Corresponding author. Tel.: q1-519-888-4567, ext. 2305; fax: q1-519-746-0614. E-mail address: [email protected] (M.M. Vijayan).

machinery. The hspyhsc70 proteins act as molecular chaperones and are crucial for protein functioning, including folding, intracellular localization, regulation, secretion, and protein degradation (Feder and Hofmann, 1999; Fink, 1999). Different genes encode for hsp70 and hsc70 and the coding sequences are continuous for hsp70, whereas for hsc70 the sequences are interrupted with several introns. The human hsc70 (Dworniczak and Mirault, 1987) and hsp70 amino acid sequences (Hunt and Morimoto, 1985) are 81% homologous with differences between the two sequences mainly attributed to stretches of one to at most five consecutive amino acid substitutions.

1532-0456/02/$ - see front matter 䊚 2002 Elsevier Science Inc. All rights reserved. PII: S 1 5 3 2 - 0 4 5 6 Ž 0 2 . 0 0 0 6 6 - 2

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In fish, hsp70s have been sequenced in different species including zebrafish (hsc70: Graser et al., 1996; Santacruz et al., 1997), tilapia (hsp70: Molina et al., 2000), medaka (hsp70 and hsc70: Arai et al., 1995), and rainbow trout (hsp70 partial sequence: Kothary et al., 1984; Zafarullah et al., 1992). The trout hsc71 and hsp70 amino acid sequences are 80% identical, whereas the human and trout hsc70 sequences are 94% identical (Zafarullah et al., 1992). In fish, there appear to be different protein isoforms for hsp70, however, no variation in protein isoforms was evident for hsc70 (White et al., 1994; Norris et al., 1995; Place and Hofmann, 2001). Like mammals, fish hsp70 expression is induced by stressors including heat shock and chemical shock, however, the stressor-induced hsc70 expression is not very clear. Hsc70 induction has been observed in response to cadmium in rat brain tumor cells (Hung et al., 1998), electrical shock in mouse brain (Kaneko et al., 1993), transient global ischemia in gerbil brain (Kawagoe et al., 1993), hypoxia in a human kidney cell line (Turman et al., 1997), and by heat shock in rat kidney cells (Sakakibara et al., 1992) or human melanoma cell lines (Dressel et al., 1998). Alternatively, decreased hsc70 expression has been observed following treatment of IB3-1 cells with sodium 4phenylbutyrate (Rubenstein and Zeitlin 2000), or mice with D-galactosamine and lipopolysaccharide (Morikawa et al., 1998). Zebrafish hsc70 mRNA expression was induced in embryos by heat shock (Santacruz et al., 1997) and during caudal fin regeneration (Tawk et al., 2000). Slightly enhanced hsc70 mRNA and protein expression was also observed after heat shock of two medaka cell lines (Arai et al., 1995), whereas heat shock did not affect hsc70 expression in topminnow hepatocytes (White et al., 1994; Norris et al., 1995). In other fish studies, no change in hsc70 mRNA expression was observed following heat shock of RTG cells (Zafarullah et al., 1992), exposure of CHSE cells to cadmium or zinc (Zafarullah et al., 1992), or exposure of rainbow trout red blood cells to azide, hypoxia or zinc (Currie et al., 1999). While most studies examined hsc70 mRNA changes, very few studies have characterized hsc70 protein expression with stressors in fish. Changes in mRNA expression do not necessarily correspond to changes in protein levels, and since proteins are functionally important, it is critical to examine hsc70 protein expression in order to understand the chap-

eroning role of these proteins in the stress tolerance process in fish. The characterization of hsc70 in fish has been limited primarily due to the lack of hsc70-specific antibody because all the antibodies currently available recognize both the inducible and the constitutive form of the protein. While mammalian hsp70 and hsc70 appear as two distinct bands on SDS–PAGE gels (Hung et al., 1998), these bands overlap with our fish samples even with 6–10% gels and 8–16% gradient gels. It is therefore, very difficult to study hsc70 expression patterns with an antibody that recognizes both hsp70 and hsc70, especially under conditions that induce hsp70, such as following heat shock or metal exposure. Our objective, therefore, was to purify hsc70 from rainbow trout and develop antibodies in order to characterize the hsc70 protein expression in rainbow trout. To this end, our two major options were to either purify rainbow trout hsc70 or to synthesize peptides to a portion of the hsc70 sequence for antibody production. Peptide sequences have been used successfully for hsp70 antibodies and total hsp70 antibodies (hsp70q hsc70), but not for hsc70 antibody generation (Dunlap and Matsumura 1997; Green et al., 1995). Indeed, human hsp70 (Hunt and Morimoto 1985) and hsc70 (Dworniczak and Mirault 1987) are ;81% homologous and the sequence differences between the two are not great enough to design a peptide sequence specific for hsc70. Consequently, we chose to purify trout liver hsc70 and used the entire protein as the antigen for antibody production. This trout-specific antibody was used as a probe to examine the expression pattern of hsc70 in response to stressors that are known to induce hsp70 in rainbow trout hepatocytes. 2. Materials and methods 2.1. Materials ATP-Agarose beads, L15 media, sodium arsenite, protease inhibitor cocktail (P2714), 2-phenoxyethanol, and Freunds Adjuvant were obtained from Sigma (St. Louis). Most general laboratory chemicals and the BCIP and NBT were from Fisher Scientific (Ontario). The 6-well Primaria plates were from Falcon (Becton Dickinson Labware, NJ). The empty Econo column and flow adaptor used for the ATP-agarose column were from Bio-Rad. All electrophoresis reagents and

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supplies, including the molecular weight markers and the secondary antibody were from BioRad. The protein A column was from Amersham Pharmacia Biotech. The total trout hsp70 antibody was from Dr E. Peter M. Candido (Biochemistry Department, UBC). Bicinchoninic acid (BCA) solution was from Pierce Chemical Co. (IL). Purified recombinant bovine hsc70 protein (SPP751) was from StressGen Biotechnologies Corp. (Victoria, BC). 2.2. Animal Rainbow Trout (Oncorhynchus mykiss) were obtained from Rainbow Springs Trout farm (Thamesford, Ont.) and were maintained in tanks with running well water (12"1 8C) under a 12-h lighty dark photoperiod. Trout were acclimated for at least 2 weeks prior to experimentation and were fed once daily to satiety (3 pt sinking food; Martin Mills Inc., Elmira, Ont). 2.3. Constitutive heat shock protein 70 (hsc70) purification 2.3.1. Arsenite treatment Fish (;300 g) were anaesthetized with an overdose of 2-phenoxyethanol (1:1000) and the liver quickly removed and placed in L15 medium on ice. The fresh livers were minced (20 ml L15 mediumyliver), transferred to a 50-ml centrifuge tube, and were slowly rocked in the presence of 50 mM sodium arsenite (NaAsO2) for 4 h at 13 8C. At the end of the 4 h, the arsenitecontaining media was replaced with fresh media and the tissue was allowed to recover overnight (typically 16–20 h) at 13 8C with gentle rocking. Following recovery, the minced liver was pelleted by centrifugation (300=g) for 5 min at 4 8C, the media removed, and the liver protein either purified immediately or stored frozen at y70 8C. 2.3.2. Sample preparation Arsenite-induced liver pieces were homogenized (4 mlyg tissue) with a PowerGen tissue homogenizer (Fisher Scientific, Ont.) in ice-cold buffer A (pH 7.5) containing Tris (20 mM), NaCl (20 mM), EDTA (0. 1 mM), MgCl2 (3 mM) and 2mercaptoethanol (15 mM) (Srivastava, 1997) and supplemented with protease inhibitor cocktail (4(2-aminoethyl)-benzenesulfonyl fluoride, trans(4-guanidino)epoxysuccinyl-L-leucylamido

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butane, bestatin, leupeptin, aprotinin and sodium EDTA; Sigma). Homogenates were centrifuged (10 000=g for 10 min followed by 100 000=g for 1 h) at 4 8C and the high-speed supernatant filtered (0.22-m syringe filter) prior to chromatography. 2.3.3. ATP-agarose affinity chromatography Lyophilized adenosine 59-triphosphate-agarose (ATP linked via carbon-8 to cross-linked 4% agarose beads) was hydrated in buffer A and a column (5.5 ml; 1=7 cm) was prepared and equilibrated. The column was fitted with a flow adaptor and attached to a Bio-Rad DuoFlow FPLC system equipped with a BioLogic QuadTec UV– Vis detector (3 mm pathlength) and conductivity detector. The filtered high-speed supernatant was loaded onto the ATP-agarose column at 0.5 mly min. The column was washed (1 mlymin) with 5 column volumes (CV) of buffer A, 10 CV buffer A supplemented with 0.5 M NaCl, and then 5 CV buffer A. The column was incubated (20 min) with buffer A supplemented with 4 mM ATP and the released proteins were eluted (0.5 mlymin). The first 2.5 CV of eluted protein were pooled and concentrated by dialysis against dry polyethylene glycol 8000 (overnight at 4 8C). The conductivity and proteinyATP absorbance (280 nm) were monitored throughout the purification. 2.3.4. Electroelution ATP-agarose-purified proteins were electrophoresed on 8% mini-gels at 200 mA for 90 min. The gels were stained with Bio-Safe Coomassie stain and the single hsc70 band from many lanes were pooled and electroeluted with a BioRad model 422 Electro-Eluter according to the manufacturer’s instructions. The hsc70 was electroeluted from the gel (7 mA, 16 h, room temperature) into ammonium bicarbonate (50 mM) buffer with SDS (0. 1% wyv). The electroeluted hsc70 was dried on low heat in a Savant Speedvac and then stored frozen at y70 8C. Approximately 15–20 mg of purified electroeluted hsc70 was recovered per gram of liver. The specificity of the purified protein was confirmed with SDS–PAGE followed by immunodetection using a trout-specific total hsp70 antibody (see below). 2.4. Antibody production Two female New Zealand white rabbits (Oryctolagus cuniculus) (4.9 and 3.9 kg) (Charles River

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Canada, Ont.) were maintained in accordance with Canadian Council of Animal Care guidelines. Lyophilized hsc70 (100 mg) was resuspended in 600 ml PBS (20 mM sodium phosphate pH 7.4, 150 mM NaCl), mixed with Complete Freunds Adjuvant (600 ml) and injected into the two rabbits, each receiving a total of 50 mg hsc70 over four initial injection spots. Three and 6 weeks after injections, blood was withdrawn and the rabbits were given booster shots (50 mg hsc70yrabbit). Protein for booster shots was prepared in the same manner as for the initial injections except Incomplete Freunds Adjuvant was used. Rabbits were exsanguinated either 9 or 12 weeks after the initial hsc70 injections. Blood collected before each injection and during exsanguination was clotted (1 h at 37 8C and overnight at 4 8C) and the serum recovered by centrifugation (10 000=g, 10 min, 4 8C). Purified hsc70 protein blots were probed with the immunized serum (and goat anti-rabbit secondary antibody) to determine the specificity of hsc70 antibodies. The serum was frozen (y 70 8C) for later purification. 2.5. Antibody purification with protein A The serum was purified with a HiTrap protein A column (5 ml) attached to a Bio-Rad DuoFlow FPLC system according to the manufacturer’s (Amersham Pharmacia Biotech.) instructions. Purified hsc70 protein blots were probed with column fractions to determine the fractions containing hsc70 antibodies. Absorbance was monitored at 280 nm and the entire eluted protein peak contained hsc70 antibody as determined by Western blotting (data not shown). Fractions containing hsc70 antibodies were pooled, aliquoted and frozen (y70 8C). 2.6. SDS–PAGE and Western blotting The tissue protein concentrations were determined by the BCA method with bovine serum albumin (BSA) as the standard. The samples were analyzed on 8% polyacrylamide gels using the discontinuous buffer system of Laemmli (1970). The gels were either stained with Bio-Safe Coomassie stain or were transferred (20 V for 30 min) onto nitrocellulose membranes with a SemiDry Transfer Unit (Bio-Rad) using transfer buffer consisting of 25 mM Tris (pH 8.3), 192 mM glycine, and 20% (vyv) methanol. Membranes were

blocked (60 min) with 5% skim milk in TBS-t (20 mM Tris pH 7.5, 300 mM NaCl, 0.1% (vyv) Tween 20) with 0.02% sodium azide. Primary and secondary antibodies were diluted in the blocking solution to the appropriate concentrations as indicated below. For total hsp70, a polyclonal rainbow trout gonadal RTG-2 antibody was used at 1:3000 dilution. This antibody recognized both the hsp70 and hsc70 in rainbow trout tissues (Forsyth et al., 1997; Vijayan et al., 1997, 1998). Our hsc70 antibody, obtained in the present study, was used at 1:3000 dilution. The secondary antibody was alkaline phosphatase-conjugated goat anti-rabbit (1:3000) antibody. The blots were incubated with primary antibody (60 min) at room temperature, washed (3=5 min) with TBS-t, incubated with secondary antibody (60 min), washed with TBS-t (2=5 min), and finally washed with TBS (1=10 min). The bands were visualized with NBT (0.033% wyv) and BCIP (0.017% wyv) and the molecular weight was visualized using prestained low range molecular weight markers (phosphorylase B 112 kDa, bovine serum albumin 81 kDa, ovalbumin 49.9 kDa, carbonic anhydrase 36.2 kDa, soybean trypsin inhibitor 29.9 kDa, lysozyme 21.3 kDa). The protein bands were scanned and the band intensities quantified using the AlphaEase software (AlphaEase Innovatech, CA). 2.7. 2D-electrophoresis Rainbow trout hepatocytes were heat shocked (q15 8C for 1 h) and then allowed to recover (13 8C for 23 h) as described below. The cells were harvested and stored frozen (y70 8C). Hepatocytes (1.5 million cells) were sonicated in 25 mM Tris (pH 7.5) with 0.4% SDS and 10 mM MgCl2. Samples were heated (90 8C for 5 min), treated with DNase (room temperature for 10 min), and the proteins were precipitated with acetone (on ice for 10 min) and collected by centrifugation (18 000=g, 3 min). Proteins (20 mgylane) were solubilized in IEF sample buffer (9.25 M urea, 1.5% pH 5–8 Bio-Lytes, 2.5% 2-mercaptoethanol, 1% triton X-100, 0.00125% bromophenol blue) and separated in the first dimension by isoelectric focussing in 5% vertical denaturing mini-slab gels with 250 ml BioLyte 5–8y10 ml of gel solution. IEF gels were electrophoresed at 150 V for 30 min and then 200 V for 5 h with 10 mM H3PO4 and 20 mM NaOH as the lower and upper electrophoresis buffers, respectively. The measured pH

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range of the IEF gel was 7.4–6.3. Proteins were separated in the second dimension by 8% SDS– PAGE. Gels were transferred for Western blotting as mentioned above.

tioned above. The proteins (30 mgylane) were separated on 8% SDS–PAGE gels and probed with trout-specific hsc70 (present study) and total hsp70 antibodies as outlined above.

2.8. Constitutive heat shock protein 70 expression in rainbow trout tissues

3. Results and discussion

Trout (;300 g) were anaesthetized with an overdose of 2-phenoxyethanol and pieces of liver, gill, heart and skeletal muscle were quickly frozen on dry ice for detection of hsc70 expression. Tissues were sonicated in 50 mM Tris (pH 7.5) with protease inhibitor cocktail (see above), centrifuged (15 000=g, 2 min), and the protein concentration determined in the supernatant as mentioned above. The proteins (60 mgylane) were separated on 8% SDS–PAGE gels and probed with hsc70 antibodies as outlined above. 2.9. Hepatocyte preparation Rainbow trout hepatocytes were isolated using collagenase perfusion according to established protocols (Sathiyaa et al., 2001) and the cells were plated in 6-well Primaria plates at a density of 1.5=106 cellsywell (0.75=106 cellsyml) in L15 media. The cells were allowed to recover overnight (13 8C) prior to experimentation. 2.10. The effect of heat shock and metal exposure on hsc70 expression Hepatocytes were heat shocked (q15 8C for 1 h) and then allowed to recover at 13 8C for up to 47 h as indicated. Samples were taken before heat shock (0 h), during heat shock (0.25, 0.5 h), immediately following heat shock (1 h) and during recovery (2, 4, 8, 24, 48 h). For comparison, parallel control samples in the absence of heat shock were also taken at 0, 1, 2, 4, 8, 24, 48 h. For metal treatment, hepatocytes were incubated with either CuSO4 (200 mM; 13 8C; 24 h), CdCl2 (10 mM; 13 8C; 24 h) or NaAsO2 (50 mM; 13 8C; 1 h then 23 h with fresh media) and preliminary studies established these dosages to be sublethal to cells. The cells were pelleted by centrifugation (13 000=g, 20 s) and stored at y 70 8C for later analysis. Hepatocytes were thawed and sonicated in 100 ml 50 mM Tris (pH 7.5) with protease inhibitor cocktail (see above) and the protein concentrations determined as men-

We have purified to homogeneity the constitutive heat shock protein 70 (hsc70) in rainbow trout and developed antibodies specific for trout hsc70. This is the first known hsc70-specific antibody in fish and allowed the characterization of hsc70 protein expression following acute heat shock and metal exposure in rainbow trout hepatocytes. 3.1. Purification of trout hsc70 A trout-specific total hsp70 antibody that recognizes both the inducible (hsp70) and the constitutive (hsc70) hsp (see Vijayan et al., 1997; Forsyth et al., 1997) was used to chart the progress of our protein purification and also to characterize the specificity of our antibody. The purification of hsc70 was carried out following established protocols using ATP-agarose affinity chromatography (Srivastava, 1997; Welch and Feramisco, 1985). Although our intention was to simultaneously purify both hsp70 and hsc70 from arsenite-treated trout liver pieces, preliminary studies confirmed that there was no increased synthesis of total hsp70 with arsenite treatment compared with the control liver pieces (data not shown). Consequently, the arsenite-treated liver homogenate was used for the purification of hsc70 protein. The majority of proteins in the high-speed supernatant obtained from arsenite-treated trout livers did not bind to the ATP-agarose affinity column and were removed by washing (Fig. 1a). There were three major bands that eluted from the ATP-agarose column (Fig. 1a) and immunodetection using trout-specific total hsp70 antibody identified the middle band as the 70-kDa heat shock protein (Fig. 1b). This protein band, from multiple lanes, was electroeluted and the protein appeared as a single band on a Coomassie-stained gel (Fig. 1a) and cross-reacted strongly with the trout total hsp70 antibody (Fig. 1b). 3.2. Generation of polyclonal and bodies for hsc70 The rabbits were immunized with the purified (electroeluted) protein and the serum from the

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Fig. 1. Hsc70 purification and antibody production. Hsc70 was purified from rainbow trout livers via differential centrifugation, ATPagarose chromatography and electroelution as detailed in Section 2. Progression of hsc70 purification was documented by (a) Coomassiestained SDS–PAGE and by (b) Western blotting with a trout-specific antibody that recognizes total hsp70 (hsp70qhsc70). The observed results are for the high speed supernatant (Sup, 40 mg) that was loaded onto the ATP-agarose column, the fraction that flowed through the column (FT, 40 mg), the concentrated eluted fraction (E, 2.25 mg), and the fraction further purified by electroelution from SDS– PAGE gels (EE, 0.7 mg for (a) and 0.28 mg for (b)). The purified electroeluted protein was used for polyclonal antibody production and the resultant rabbit serum was purified by protein A chromatography. (c) A representative Western blot showing that the purified antibody cross-reacts with the ATP-eluted fraction (lane 1, 2.25 mg) and purified recombinant bovine hsc70 (lanes 2 and 3, 2 mg). Parallel detection under the same conditions as with the purified antibody revealed no detectable labeling of purified recombinant bovine hsc70 with the trout-specific total hsp70 antibody (lanes 4 and 5, 2 mg). (d) Hsc70 and hsp70 were separated by 2D electrophoresis from heat-shocked (q 158 HS 1 h followed by 23 h recovery at ambient) trout hepatocytes and the proteins were visualized by Western blotting with either the purified hsc70 antibody, the trout-specific total hsp70 antibody or a combination of both these antibodies mixed together. The dark arrow and hollow arrow indicates the major and minor hsc70 spots, respectively; the thin arrow indicates hsp70 spot.

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Fig. 2. Hsc70 expression in rainbow trout tissue. Trout heart, gill, skeletal muscle and liver were excised and immediately frozen on dry ice. Samples (60 mg) were separated by 8% SDS–PAGE, transferred to a nitrocellulose membrane and probed with trout-specific hsc70 antibody. Tissues were taken from two fish.

immunized rabbits was partially purified by protein A affinity chromatography. The antibody crossreacted strongly with the ATP-eluted fractions confirming that the antibodies were specific to the purified protein (Fig. 1c). We further confirmed the specificity of our hsc70 antibody by probing purified recombinant bovine hsc70. Our antibody showed clearly very high cross-reactivity with bovine hsc70, whereas the total hsp70 antibody showed no cross-reactivity (Fig. 1c) providing further evidence that our antibody is highly specific to hsc70 with much stronger cross-reactivity for hsc70 than the total hsp70 antibody. Despite the substantial purification achieved with the protein A column, some minor vertical streaks were still observed when using the antibody for Western blotting (Figs. 1c, 2, 3d). The non-specific streaks did not disappear with either ammonium sulfate precipitation or drying followed by reconstitution of the antibody, although further dilution of the antibody did slow the appearance of streaks. Interestingly, once the antibody solution was used to probe a number of blots, the streaks and nonspecific binding disappeared. The antibody was very stable at 1:3000 dilution in 5% skim milk (with sodium azide) and the diluted antibody could be used to probe numerous blots for many months. Our hsc70 antibody also recognized one and sometimes two faint bands of approximately 55–65 kDa in the ATP-agarose-purified fraction (Figs. 1c, 3d) suggesting that these bands were ATPbinding proteins. The electroeluted purified hsc70 used for antibody production was a single band and contained no detectable 55–60-kDa proteins (Fig. 1a). A likely explanation is that the lower bands may contain epitopes similar to those of hsc70 or perhaps the antibody was detecting a breakdown product of hsc70.

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The specificity of the hsc70 antibody was further assessed by 2D-gel electrophoresis and Western blotting. While mammalian hsp70 and hsc70 appear as two distinct bands on SDS–PAGE gels (Hung et al., 1998), these bands overlap with our fish samples even with 6–10% gels and with 8– 16% gradient gels. However, we were able to separate hsc70 and hsp70 by 2D-gel electrophoresis of heat-shocked hepatocytes and Western blotting with the hsc70 antibody showing a prominent spot (dark arrow) with a pI more acidic than hsp70 (thin arrow) (Fig. 1d). This agrees with other studies that showed hsc70 to be more acidic than hsp70 in fish (Norris et al., 1995). Also, our antibody also labeled a minor spot with a pI that was more basic than the prominent hsc70 spot (hollow arrow; Fig. 1d). The total hsp70 antibody showed a large streak (Fig. 1d) that may be due to the fact that the antibody recognized both hsc70 and hsp70 and also because of the presence of multiple hsp70 isoforms with slightly different pI values (Norris et al., 1995). However, alignment of the two blots (probed with hsc70 and total hsp70 antibody) showed some overlap of spots and it is not clear if the minor more basic spot labelled with the hsc70 antibody represents an additional hsc70 isoform or is labeling hsp70. The observed lack of hsc70 induction with heat shock, copper, cadmium, and arsenite (Figs. 3 and 4) strongly suggests that the hsc70 antibody is recognizing at least two separate hsc70 isoforms in rainbow trout. 3.3. Tissue distribution of hsc70 The constitutive member of the hsp70 family of proteins, hsc70, plays an important role as molecular chaperone in unstressed cells (see Fink, 1999). Recently, hsc70 was purified from the white muscle of Goby and was shown to exhibit chaperoning function at temperature ranges far above their physiological tolerance (Place and Hofmann, 2001) suggesting a role for this protein in the heat shock response in fish tissue. Using our antibody we were able to detect hsc70 in different tissues, including liver, heart, gill and white muscle of rainbow trout (Fig. 2). The lower hsc70 content in the skeletal muscle may be due to the lower

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Fig. 3. The effect of heat shock on hsc70 protein expression. (A,B) Hepatocytes were heat shocked for 1 h (q15 8C) and sampled following recovery at 3 (lane 2) or 23 h (lane 3) at ambient temperature (13 8C). Control hepatocytes (lane 1) were sampled just prior to stressor exposure. Samples (30 mg) were probed with either trout-specific purified hsc70 antibody (a) or trout-specific total hsp70 antibody (b). The experiment was repeated four times with similar results. The temporal changes in hsc70 protein expression was further studied with three separate fish over a longer period of time and samples were probed with trout-specific hsc70 antibody. (c) A representative Western blot of control cells in the absence of heat shock and sampled at 0, 1, 2, 4, 8, 24, 48 h. (d) A representative Western blot of heat-shocked hepatocytes (1 h at q15 8C) and sampled either during the heat shock (0.25, 0.5 h), immediately after heat shock (1 h) or during recovery from heat shock (2, 4, 8, 24, 48 h). (e) Hsc70 expression prior to heat shock (0) and at 48 h either in the absence (48yHS) or presence of heat shock (48qHS); values represent mean"S.E.M. (ns3); there was no statistically significant difference (P-0.05, paired t-est).

protein turnover in this tissue compared to liver, gill and heart. Studies are underway to characterize the temporal pattern of hsc70 expression in various trout tissues in response to different stressors, including heat and chemical shock.

3.4. The effect of heat shock on hsc70 protein expression in trout hepatocytes Heat shock (HS; q15 8C) exposure induced total hsp70 protein accumulation by approximately

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Fig. 4. The effect of metals on hsc70 protein expression. Rainbow trout hepatocytes were incubated either in the absence (Control; Con) or presence of copper (Cu; 200 mM CuSO4), cadmium (Cd; 10 mM CdCl2) or arsenite (As; 50 mM NaAsO2) as outlined in Section 2. Samples (30 mg) were separated by 8% SDS–PAGE, transferred to nitrocellulose membrane, and probed with purified troutspecific hsc70 antibody (a,c) or trout-specific total hsp70 antibody (b,d). All blots are representative of three separate fish. (e) Hsc70 protein expression with metals were quantified and shown as mean"S.E.M. (ns3 fish); there was no statistically significant difference (P-0.05; paired t-est).

fivefold over a 24-h period in trout hepatocytes in primary culture (Fig. 3b) and this result concurred with a recent study showing a similar increase in hsp70 response with HS in trout hepatocytes (Boone et al., 2002). When a parallel blot to that shown in Fig. 3b was probed with our purified trout-specific hsc70 antibody, no increase in hsc70 protein expression was observed (Fig. 3a). Also, there were no temporal changes in hsc70 protein expression either in the absence of heat shock (Fig. 3c) or during heat shock and the recovery period (Fig. 3d,e). These results concur with other studies showing a similar lack of change in hsc70 expression (35S-labelling and 2D-electrophoresis)

with heat shock in fish hepatocytes (White et al., 1994; Norris et al., 1995). 3.5. The effect of metal exposure on hsc70 protein expression Heavy metal exposure results in higher hsp70 expression in fish tissues, including hepatocytes (Iwama et al., 1998; Boone et al., 2002). Our results clearly show elevated hsp70 accumulation (three to sixfold) following exposure of trout hepatocytes to either CuSO4, CdCl2 or NaAsO2 (Fig. 4b,d). However, we show for the first time that hsc70 protein expression is not altered by

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metal exposure in fish cells (Fig. 4a,c,e). This lack of higher hsc70 expression with metals in trout hepatocytes (Fig. 4) may not be due to altered protein turnover because previous studies also failed to observe any changes in hsc70 mRNA accumulation upon exposure of CHSE cells to cadmium or zinc (Zafarullah et al., 1992), or exposure of rainbow trout red blood cells to azide, hypoxia or zinc (Currie et al., 1999). Taken together, these results suggest that hsc70 expression is not modulated by sublethal acute stressors in fish. However, although the hsc70 content does not change with stressors, intracellular partitioning of hsc70 content may be playing a key role in allowing cells to cope with stress. Indeed nuclear translocation of hsc70 was shown to be important in allowing HeLa cells to cope with heat shock (Chu et al., 2001). The availability of trout-specific hsc70 antibody will, therefore, allow us to characterize the stressor-induced intracellular trafficking of hsc70 in fish cells and its implication in the cellular stress response process. Acknowledgments This study was supported by the Natural Sciences and Engineering Research Council (NSERC), Canada, operating grant to M.M. Vijayan. The authors would like to thank Dr P. Candido for the generous gift of the trout-specific total hsp70 antibody and Mr M. Ryan for assistance with the generation of hsc70 antibodies. References Arai, A., Naruse, K., Mitani, H., Shima, A., 1995. Cloning and characterization of cDNAs for 70-kDa heat-shock proteins (Hsp70) from two fish species of the genus Oryzias. Jpn. J. Genet. 70, 423–433. Boone, A.N., Ducouret, B., Vijayan, M.M., 2002. Glucocorticoid-induced glucose release is abolished in trout hepatocytes with elevated HSP70 content. J. Endocrinol. 172, R1–R6. Chu, A., Matusiewicz, N., Stochaj, U., 2001. Heat-induced nuclear accumulation of hsc70s is regulated by phosphorylation and inhibited in confluent cells. FASEB J. 15, 1478–1480. Currie, S., Tufts, B.L., Moyes, C.D., 1999. Influence of bioenergetic stress on heat shock protein gene expression in nucleated red blood cells of fish. Am. J. Physiol. 276, R990–R996. Dressel, R., Johnson, J.P., Gunther, E., 1998. Heterogeneous patterns of constitutive and heat shock induced expression of HLA-linked HSP70-1 and HSP70-2 heat shock genes in human melanoma cell lines. Melanoma Res. 8, 482–492.

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