2019 Volume 84 Issue 4 Pages 299-308
Heat shock proteins (Hsps) are molecular chaperones that respond to various external and internal cellular stresses. Hsp90 is an abundant cellular heat shock protein. It has two isoforms, Hsp90α and Hsp90β. In fish, expression of the isoforms is augmented by various stress signals, including thermal change, salinity, pH, ammonia, infection, and environmental pollutants. In this study, we isolated both isoforms of Hsp90, designated rkHsp90α and rkHsp90β, from the liver of Kumgang fat minnow, Rhynchocypris kumgangensis, a small freshwater fish that is endemic to Korea. Sequences of the isolated isoforms showed homology with the corresponding isoforms of teleost Hsp90. Both isoforms of rkHsp90 were highly expressed in the liver compared to other tissues, including brain, gastrointestinal tract, gills, and muscle. Water temperature elevation induced increased hepatic and muscular expression of rkHsp90α, but not rkHsp90β. Both isoforms did not respond to lipopolysaccharide challenges. Exposure to environmental pollutants promoted expression of rkHsp90β, but not rkHsp90α. The collective findings support the proposal that rkHsp90α and rkHsp90β act as molecular chaperones that respond to distinct cellular stresses. Both could serve as useful biomarkers for assessing cellular stress in Kumgang fat minnow.
Heat shock proteins (Hsps) play important roles during internal and external cellular stress. Hsps are found in various cellular compartments in eukaryotic and prokaryotic cells. These proteins are categorized into several families and named according to their function, sequence homology, and molecular mass. Families primarily include Hsp100, Hsp90, Hsp70, Hsp60, and small Hsp. Hsps function as chaperone molecules in response to thermal stress, ischemia, protein degradation, microbial infection, and exposure to pollutants (Roberts et al. 2010).
The Hsp90 family is one of the most abundant protein families in eukaryotic cells, comprising 1–2% of cellular proteins under non-stress conditions. It has a molecular weight of approximately 90 kDa and comprises several members, depending on their localization. Hsp90α and Hsp90β are found in the cytoplasm. A 94-kDa glucose-regulated protein and tumor necrosis factor receptor-associated protein-1 are localized in the endoplasmic reticulum and mitochondria, respectively (Hoter et al. 2018). Although the cytoplasmic Hsp90α and Hsp90β proteins are structurally and functionally similar, they have distinctive properties. The expression of Hsp90α is inducible, while Hsp90β is expressed constitutively. There are also differences in the dimerization of these two isoforms. Both isoforms show specificity in cell differentiation and development (Sreedhar et al. 2004).
The expression level of Hsp90 is a useful biomarker for cellular stress in various organisms. In fish, gradual and acute temperature increase and bacterial infection induce the up-regulation of Hsp90 expression (Pu et al. 2016, Rebl et al. 2018). The expression levels of Hsp90 are also elevated in water contaminated with heavy metals (Mihailovic et al. 2016). Hsp90 is also used as a health assessment biomarker associated with oxidative stress and exposure to residual pesticides (Miller et al. 2015, Xing et al. 2015).
The Kumgang fat minnow (R. kumgangensis) is a small freshwater fish belonging to the family Cyprinidae. It is a representative cold-water fish and is endemic to Korea. Their survival may be threatened by various environmental factors, such as global warming and pollution. Thus, it is important to monitor physiological stresses in the fish using biomarkers. In previous studies, we isolated the gene encoding the warm-temperature acclimation-associated 65-kDa protein (Wap65) in Kumgang fat minnow and assessed its value as a biomarker. The gene responded effectively to cellular stresses induced by temperature increase, bacterial infection, and exposure to environmental pollutants (Im et al. 2016, Kwon and Ghil 2017). Although we have previously isolate Wap65 genes, it is necessary to obtain more gene for the accurate evaluation of cellular stress on the fish. Therefore, we have identified Hsp90 genes that have been reported to be responded in various cellular stresses in many organisms.
In the present study, we isolated two isoforms of Hsp90 from the liver of Kumgang fat minnow. They were designated rkHsp90α and rkHsp90β. Both genes were cloned using a polymerase chain reaction (PCR)-based strategy, and their sequences were compared with the corresponding isoforms of teleost Hsp90 genes. We employed real-time quantitative PCR (RT-qPCR) to determine tissue distribution profile of the isoforms and their expression levels induced by cellular stresses that included water temperature elevation, inflammation, and exposure to environmental pollutants.
The methods used for fish collection and water temperature control have been described previously (Im et al. 2016, Kwon and Ghil 2017). Briefly, adult Kumgang fat minnows (average length 7.39±0.52 cm, weight 3.25±0.89 g) were collected from Hwacheon-gun and Inje-gun, Gangwon-do, Republic of Korea. They were transferred to a 60-L fish tank equipped with circulatory and cooling systems in the laboratory and fed twice a day with commercial feed (Bio-Pure FD blood worms; Kyorin Co. Ltd., Japan). In all experiments, the animals were not fed for 2 days before tissue collection. After collection, the fish were acclimated at the same temperature at which the fish were collected (11°C), with a 12 : 12 light : dark photoperiod for 3 weeks.
Isolation of complementary DNA (cDNA) for rkHsp90α and rkHsp90βWe isolated both isoforms of rkHsp90 cDNA from the liver of Kumgang fat minnow as described previously (Im et al. 2016, Kwon and Ghil 2017). Briefly, total RNA was isolated by Easy-spin (DNA free) Total RNA Extraction Kit (Intron Biotechnology, Daejeon, Korea) and reverse-transcribed by AccuPower RT PreMix (Bioneer, Daejeon, Korea). We isolated the rkHsp90 genes by dividing three fragments; the middle-, 5′- and 3′-fragment. The middle-fragment (598–1161 bp in rkHsp90α and 913–1974 bp in rkHsp90β) of genes was amplified by PCR using the resulting cDNA and the rkHsp90α-M and rkHsp90β-M primer sets (Table 1). The 5′-fragments were obtained with the aid of an additional PCR reaction using cDNA and the rkHsp90α-5′ and rkHsp90β-5′ primer sets. To obtain the 3′-fragment, the 3′-rapid amplification of cDNA ends (3′-RACE) technique was used with cDNA and the rkHsp90α-3′-RACE and rkHsp90β-3′-RACE primer sets, using 3′-full RACE Core Set (TaKaRa-Bio Inc., Otsu, Japan) according to the manufacturer’s instructions. All primers were designed to target highly conserved regions based on reference to known sequences in other fish (Table 1). PCR was performed using the Veriti 96-well Thermal Cycler (Applied Biosystems, Foster City, CA, USA). The amplified fragments were inserted into pTop Blunt V2 plasmids (Enzynomics, Daejeon, Korea) and the sequences were obtained via sequence analysis of the plasmid.
| Primer | Sequence |
|---|---|
| rkHsp90α-M | 5′-GAC AAG GAG GTG AGC GAC GAT GAG-3′ |
| 5′-CCG TYT CRA ACA GCA GRA K-3′ | |
| rkHsp90α-5′ | 5′-GCG CTA GTT ACG GAT CTC TCA AAG-3′ |
| 5′-AGC TGG CTC ATC TTC CTT GT-3′ | |
| rkHsp90α-3′-RACE | 5′-GAA ATC AAC CCT CTC CAT CCC-3′ |
| rkHsp90β-M | 5′-GGA GAG TTT TAC AAG AGC CTG ACC-3′ |
| 5′-GAC CAG ATC CTT CAC GGC TTT ATC-3′ | |
| rkHsp90β-5′ | 5′-AAC CAA GAT GCC TGA AGA AAT GCG-3′ |
| 5′-TCA GAG TCA ACC ACA CCA CGG ATG-3′ | |
| rkHsp90β-3′-RACE | 5′-GGC AAA GAA ACA CCT GGA GA-3′ |
| rkHsp90α RT-qPCR | 5′-GCG CTA GTT ACG GAT CTC TCA AAG-3′ |
| 5′-AGC TGG CTC ATC TTC CTT GT-3′ | |
| rkHsp90β RT-qPCR | 5′-GCG CTA GTT ACG GAT CTC TCA AAG-3′ |
| 5′-AGC TGG CTC ATC TTC CTT GT-3′ | |
| rk-β-actin RT-qPCR | 5′-CTT GAC TTT GAG CAG GAG ATG G-3′; |
| 5′-CAA GAA GGA TGG CTG GAA CA-3′ | |
| rkGAPDH RT-qPCR | 5′-GAG GCT GGG ATG ATG TTC TGA CTG-3′ |
| 5′-ATG AGC ACT GTT CAC GCT ATC ACA-3′ |
The Basic Local Alignment Search Tool (BLAST) of sequence analysis program (http://www.ncbi.nlm.nih.gov.BLAST) was employed for comparison of rkHsp90 isoforms with published sequences in GenBank. Phylogenetic tree analyses were conducted according to the amino acid sequences of the full-length Hsp90 using the BioEdit and MEGA6 program. The stability of the phylogenetic trees was evaluated as 10000 bootstrapping replications. The GenBank accession numbers of amino acids sequences are listed in Supplementary Table S1. Analysis of the three-dimensional (3D) structure of rkHsp90α and rkHsp90β was performed by iterative threading assembly refinement (I-TASSER) online web server (http://zhanglab.ccmb.med.umich.edu/I-TASSER). Structures were presented by a carton diagram using PyMol software.
High-temperature treatment and tissues samplingTo determine the tissue distribution of rkHsp90 isoforms, we isolated various tissues, including brain, gastrointestinal (GI) tract, gill, liver, and muscle from fish acclimated in control temperature, 11°C. For the water temperature elevation experiment, the fish were divided into control and experimental groups. The water temperature of the control group was maintained at 11°C until tissue harvest and the experimental group was increased by 1.0±0.5°C h−1 over 8 h (from 11 to 19°C) and subsequently maintained at 19°C for the indicated times (0 h, 1 h, 6 h, 12 h, 24 h, 1 w and 2 w). At the conclusion of the water temperature elevation experiment, fish were sacrificed by an overdose of MS222 anesthetic (360 mg L−1, Sigma-Aldrich, St. Louis, MO, USA) for 10 min. Five fish were used at each sampling point. All animal experiments were conducted in keeping with the relevant national and international guidelines.
Lipopolysaccharide (LPS) exposureThe fish were anesthetized by exposure to 360 mg L−1 of MS222 for 3 min, followed by intraperitoneal injection with 0.1 mL of phosphate-buffered saline (PBS) as the control group or Escherichia coli 0111: B4 LPS (Sigma-Aldrich) dissolved in PBS (5 µg g−1 body weight) as the experimental group. After 48 h, the liver was harvested from each fish. During the experiment, the water temperature was set at 11°C, and six fish were used for each condition.
Exposure of environmental pollutantsFor heavy metal exposure, CdCl2 (Cd, Sigma-Aldrich), CuCl2·2H2O (Cu, Sigma-Aldrich), or FeCl3·6H2O (Fe, Sigma-Aldrich) was dissolved at a concentration of 5 µg L−1 in the fish tank and incubated the fish for 48 h. Six fish were used for each condition. Bisphenol A (BPA, Sigma-Aldrich) or estradiol (Sigma-Aldrich) was added to the fish tank to a concentration of 500 µg L−1 or 500 ng L−1, respectively, for 48 h or 96 h. Twelves fish were used for each condition. After exposure to the environmental pollutants, the liver was isolated from each fish. The water temperature of fish tank was set at 11°C.
RT-qPCR analysisThe mRNA expression levels of the rkHsp90 isoforms were analyzed with the rkHsp90α-RT-qPCR and rkHsp90β-RT-qPCR primer sets (Table 1). Two primer sets of internal controls, rkβ-actin-qRT-PCR, and rkGAPDH-qRT-PCR for β-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), respectively, were used to normalize the rkHsp90 mRNA expression levels. One microliter of cDNA synthesized from total RNA (1 µg) using TOP Real qPCR 2×Premix (Enzynomics) and 10 pmol of each primer was combined to make the 20 µL PCR reaction mixture. PCR reactions were performed by CFX96 system (Bio-Rad Laboratories, Hercules, CA, USA) with 95°C for 10 min (initial denaturation), followed by 40 cycles of 95°C for 10 s, 60.8°C for 30 s, and 72°C for 30 s. Relative mRNA expression levels were quantified after normalizing values to those of two internal control genes using CFX Manager software (Bio-Rad Laboratories) following the manufacturer’s instructions. All RT-qPCR experiments are representative of at least three independent experiments performed in triplicate. Statistical differences were evaluated as Student’s unpaired t-test using the SigmaPlot 10.0 software program.
To isolate rkHsp90α and rkHsp90β isoforms from Kumgang fat minnow, we used a PCR-based cloning strategy with the primer sets presented in Table 1. The isolated rkHsp90α and rkHsp90β cDNA included a 2106 and 2190 bp open reading frame, respectively, beginning with the ATG codon and ending with a TGA and TAA stop codon, respectively (Supplementary Fig. S1). The rkHsp90α cDNA exhibited 75% homology with rkHsp90β. The rkHsp90α cDNA showed the following homologies with Hsp90α from other teleost fish and human: Danio rerio, 81%; Esox lucius, 80%; Megalobrama amblycephala, 80%; Salmo salar, 79%; Astyanax mexicanus, 76%; and Homo sapiens, 71%. The rkHsp90β cDNA showed the following homologies with Hsp90α from other teleost fish and human: Cyprinus carpio, 97%; Gobiocypris rarus, 96%; Megalobrama amblycephala, 96%; Tanichthys albonubes, 94%; Danio rerio, 93%; and Homo sapiens, 80%.
The open reading frame of rkHsp90α and rkHsp90β was predicted to encode a 701 and 729 amino acid protein, respectively, containing major ATP/geldanamycin binding site (asterisk in Fig. 1) and phosphorylation domain for casein kinase II (arrowhead in Fig. 1); five Hsp90 conserved motifs (open box in Fig. 1); and a C-terminal dimerization domain MEEVD sequence (light-gray shaded box in Fig. 1). The rkHsp90α amino acid sequence displayed 78% homology with rkHsp90β. The rkHsp90α amino acid sequence showed following the homologies with Hsp90α from other teleost fish and human: A. mexicanus, 84%; M. amblycephala, 84%; E. lucius, 83%; D. rerio, 82%; S. salar, 81%; and H. sapiens, 82%. The rkHsp90β amino acid sequence showed the following homologies with Hsp90β from other teleost fish and human: C. carpio, 99%; G. rarus, 99%; M. amblycephala, 99%; T. albonubes, 98%; D. rerio, 97%; and H. sapiens, 90%. We generated a phylogenetic tree of the Hsp90 to analyze the rkHsp90α and rkHsp90β amino acid sequences in the larger context of vertebrate Hsp90 (Fig. 2). They were more closely related to teleost Hsp90 than to human homologues.
We next predicted and compared 3D structure of rkHsp90 proteins using I-TASSER protein structure prediction algorithm (http://zhanglab.ccmb.med.umich.edu/I-TASSER/). As shown in Fig. 3a and b, the overall 3D structures of both rkHsp90 proteins were similar. The tertiary structure of both isoforms was modeled with high confidence value (C-score of −0.06 and 0.33 for rkHsp90α and rkHsp90β, respectively) and a TM-score of 0.71±0.12 for rkHsp90α and 0.76±0.10 for rkHsp90β, indicating a model of correct topology.
To determine the mRNA expression level of rkHsp90α and rkHsp90β in various tissues, including brain, GI tract, gill, liver, and muscle in Kumgang fat minnow, we performed RT-qPCR analysis (Fig. 4). Among the tested tissues, mRNA expression levels of both isoforms were highest in the liver and lowest in muscle. The rkHsp90α and rkHsp90β mRNA level in the liver was approximately 106.9- and 21.9-fold higher, respectively, than that in the muscle.
To investigate whether the mRNA expression levels of both rkHsp90 gene isoforms are affected by high water temperature, the water temperature was increased from 11°C (control temperature) to 19°C (experimental temperature) by 1±0.5°C h−1 for 8 h (Fig. 5). While rkHsp90α mRNA expressions were significantly increased in the GI tract, liver, and muscle, rkHsp90β expression was not altered by a water temperature increase in all tested tissues (Fig. 5a–e). The exposure to the high water temperature significantly decreased the expression of rkHsp90α in the GI tract after 6 h and 24 h, and rkHsp90β in the gill after 12 h, 1 w, and 2 w.
Next, we determined the effect of bacterial infection on the expression of rkHsp90 genes after intraperitoneal injection of bacterial LPS (Fig. 6). LPS injection did not induce rkHsp90 expression but instead reduced the expression of rkHsp90 isoforms significantly.
Finally, we investigated whether the expression levels of rkHsp90 isoforms were affected by exposure to environmental pollutants (Fig. 7). Exposure to cadmium, copper, iron, BPA, and estradiol significantly increased the expression of rkHsp90β, but not rkHsp90α, except for the 48-h exposure to estradiol. The expression of rkHsp90α was significantly decreased by 96 h exposures to BPA and estradiol.
In this study, we cloned Hsp90α and Hsp90β isoforms from Kumgang fat minnow liver and investigated their expression profiles in response to several cellular stresses, including water temperature increase, bacterial infection, and exposure to environmental pollutants. Both isoforms displayed high amino acid sequence homology and similar 3D structures and were highly expressed in the liver. Expression of rkHsp90α was generally sensitive to water temperature elevation, and rkHsp90β was responded to exposure to environmental pollutants.
The deduced amino acid sequences of rkHsp90α and rkHsp90β had extensive homology with that of teleost and human Hsp90α and Hsp90β, respectively. Moreover, the open reading frame of rkHsp90α and rkHsp90β was found to contain five Hsp90 conserved motifs. The third rkHsp90 conserved motif contains GxxGxG, which is necessary for binding ATP (Prodromou et al. 1997). The consensus sequence MEEVD at the C-terminus of rkHsp90 is strictly conserved and shared with the most members of the Hsp90 family (Zhou et al. 2017). This sequence is involved in dimerization of Hsp90 proteins and also functions as a binding motif to other proteins that contain multiple copies of the tetratricopeptide repeat (Donnelly and Blagg 2008, Blundell et al. 2017). These findings suggest that rkHsp90α and rkHsp90β possess the major structural and functional domains found in typical Hsp90 (Gupta 1995).
Although Hsp90 isoforms are known to be ubiquitously expressed in various tissues, there are also evidence of tissue-specific expression. Hsp90 isoforms are mainly expressed in liver and brain in various fish (Liu et al. 2012, Ni et al. 2014, Sun et al. 2015, He et al. 2016, Zhou et al. 2017). In the pond loach (Misgurnus anguillicaudatus), Hsp90α and Hsp90β are highly expressed in the brain and kidney, respectively (Yan et al. 2017). The kidney is the main tissue for the expression of hsp90β in Schizothorax prenanti (Pu et al. 2016). Several lines of evidence also indicate that Hsp90 gene is highly expressed in the digestive tract (Huang et al. 2014, Peng et al. 2016) or gonad (Fu et al. 2011, Liu et al. 2012, 2015), suggesting that Hsp90 can have a tissue-specific expression pattern depending on the species. In this study, rkHsp90 was highly expressed in the liver and brain, suggesting that rkHsp90 elicits cytoprotective function in these tissues that are necessary for the adaptation of cells to environmental stress.
Hsp90α expression is generally lower compared to Hsp90β in most cells (Sreedhar et al. 2004). In mammals, Hsp90α expression is highly inducible when the cells are in an unusual condition, such as tumor progression and enhanced cell cycle regulation (Yufu et al. 1992, Jerome et al. 1993). In contrast to Hsp90α, Hsp90β expression is thought to be constitutive and is probably involved in long-term cellular adaptation. Hsp90β expression is associated with the development of drug resistance (Sreedhar et al. 2003). Hsp90β is the major form of Hsp90 involved in normal cellular functions.
Hsp proteins protect cells from stressors by preventing the irreversible loss of vital proteins and facilitating their subsequent regeneration (Molina et al. 2002, He et al. 2010). Hsp90 responds to various stressors, including thermal change, infection, salinity, hypoxia, hydrogen concentration, and environmental pollutants. Water temperature is one of the most influential environmental factors that affect the distribution, abundance, physiology, and behavior of fish. In this study, temperature elevation induced an increase in the expression of rkHsp90α, but not rkHsp90β, which is coincident with results of previous studies comparing two isoforms in fish (Fangue et al. 2006, Manchado et al. 2008, Jeronimo et al. 2017). In a study of each isoform, Hsp90α expression generally responds to thermal stress (Huang et al. 2014, Zhou et al. 2017) but the expression of Hsp90β depends on species and tissues (Peng et al. 2016, Yan et al. 2017). The collective findings support the idea that rkHsp90α expression is inducible and increased by thermal stress, while the expression of rkHsp90β is constitutive and stable. Hsp90 expression is induced by heat shock factor which mediates gene transcription of Hsp90 by binding to its promoter (Roberts et al. 2010, Wang et al. 2011). Further studies are needed to clarify the roles of a heat shock factor in the expression of rkHsp90α and rkHsp90β during thermal stress.
In general, Hsp90 expression responds to bacterial infection (He et al. 2016, Pu et al. 2016, Song et al. 2018). Similar to the temperature response, the expression of Hsp90 in response to infection displays tissue- and isoform-specificity. Bacterial infection in Siniperca chuatsi results in markedly up-regulated expression of Hsp90α in kidney and spleen tissue and mild up-regulated expression of Hsp90β in spleen tissue while suppressing Hsp90β in the kidney (Wang et al. 2016). In Channa argus, Hsp90 expression is up-regulated by bacterial challenge in kidney, muscle and spleen tissue, while expression is down-regulated in liver tissue (Zhou et al. 2017). Hsp90 expression also displays pathogen specificity. In Ictalurus punctatus, Flavobacterium columnare infection generally induces increased Hsp90 expression, while Edwardsiella ictaluri infection is not significantly regulated or is not altered at all (Xie et al. 2015). Hsp90 is also reportedly involved in the initial period of bacterial infection, with its expression being increased during early period of infection and down-regulated subsequently. In Labeo rohita, the Hsp90 gene is up-regulated in liver tissue during the first 6 h of bacterial infection but is down-regulated from 24 to 72 h of infection (Das et al. 2015). In M. amblycehala, Hsp90 expression is increased up to 4 h after the establishment of bacterial infection, with no change in expression thereafter (Ding et al. 2013). This fluctuating pattern of Hsp90 expression has been described previously (Araya et al. 2010, Rungrassamee et al. 2010). Presently, we did not observe an increase in the expression of rkHsp90 by LPS challenge in the liver of Kumgang fat minnow. Therefore, it is possible that expression of Hsp90 in response to LPS is tissue-, pathogen-specific, and/or that the up-regulation of the Hsp90 gene occurs early in infection (during the first 24 h). Further studies are needed to determine Hsp90 expression in other tissues and during the early stage of infection.
The effects of heavy metals on Hsp90 expression in fish have been studied. Hsp90 expression can change in response to heavy metals depending on species and tissues. Liu and colleague cloned Hsp90 gene in T. albonubes and determined that its expression was decreased by copper and cadmium exposure (Liu et al. 2012). Exposure to copper and cadmium induces a significant increase in Hsp90 expression in C. carpio (Jiang et al. 2016). In L. rohita, cadmium exposure leads to the up-regulation of Hsp90 (Giri et al. 2016). In another study, cadmium exposure augmented the expression of Hsp90α, but not Hsp90β (Hermesz et al. 2001). However, in Morone saxatilis, copper did not alter the Hsp90 expression level. Other heavy metals, such as zinc and lead, are also associated with the up-regulation of Hsp90 expression (Moniruzzaman et al. 2017). Presently, exposure to cadmium, copper, and iron-induced increased expression of rkHsp90β but not rkHsp90α, suggesting that rkHsp90β might be a useful candidate biomarker for monitoring heavy metal-mediated aquatic pollution.
Heat shock proteins play important roles in cellular stress induced by environmental estrogen in various organisms including insects and mammals (Papaconstantinou et al. 2003, Lee et al. 2006). Environmental estrogens also appear to affect Hsp90 expression in fish. Treatment with atrazine, which causes endocrine disruption, up-regulates Hsp90 and estrogen receptor gene expression (Yang et al. 2010). Application of the potent synthetic estrogen 17α-ethynylestradiol induces vitellogenin production and this activity is reduced by Hsp90 inhibitor treatment, suggesting the involvement of Hsp90 in ER-mediated cellular signaling (Osborne et al. 2007). Our observations that BPA and estradiol generally up-regulate rkHsp90β expression additionally suggest the involvement of rkHsp90β in ER-mediated signaling, including increment of receptor stability.
In conclusion, we isolated both rkHsp90α and rkHsp90β isoforms from Kumgang fat minnow liver tissues. These isoforms are highly homologous to corresponding isoforms of teleost Hsp90 and are predominantly expressed in the liver tissue. Temperature elevation induces an increase in rkHsp90α in liver and muscle tissues. In contrast, hepatic rkHsp90β expression responds to heavy metal and environmental estrogen treatments. We propose that both rkHsp90 isoforms function as chaperones for cellular stress-mediated proteins. The isoforms may be valuable as biomarkers for assessing cellular stress in Kumgang fat minnow.
With Supplementary files of Table S1 and Fig. S1.
This work was supported by Kyonggi University Research Grant 2016.
