Stress Protein HSP70 in Fish Abstract e-mail:

Aqua-BioScience Monographs Vol. 3, No. 4, pp. 111–141 (2010)
www.terrapub.co.jp/onlinemonographs/absm/
Stress Protein HSP70 in Fish
Michiaki Yamashita,* Takeshi Yabu and Nobuhiko Ojima
National Research Institute of Fisheries Science
2-12-4 Fukuura, Yokohama 236-8648, Japan
e-mail: [email protected]
Abstract
Stress proteins (heat-shock proteins, HSPs), which comprise an evolutionally wellconserved protein family, are induced in response to a variety of stress conditions and
metabolic insults. When cells are subjected to sudden environmental changes, stress proteins are induced and play a central role in cellular homeostasis. A response to sudden
adverse environmental changes is referred to as the heat-shock or stress response and is
accompanied by a rapid increase in the synthesis of stress proteins. Given the importance
of stress proteins in thermal adaptation at the cellular level, we have studied the expression, regulation, and protective functions of the members of the HSP70 stress protein
family under normal and stress conditions in a variety of fish species. HSP70/heat-shock
cognate protein-70 (HSC70) plays essential roles in the receptor complex formation and
activation of Activin/Nodal/transforming growth factor-β and bone morphogenetic protein receptors and facilitates Nodal signaling. In addition, chaperone-mediated autophagy
assisted by HSP70/heat-shock cognate (HSC)70 may be responsible for the stress responses in fish cells. HSP70 and HSC70 translocated into the lysosomes were found to
accelerate protein degradation and catabolism under both stressed and normal conditions.
1. Introduction
,
Stress responses, such as the temperature-dependent
regulation of gene expression, enable marine organisms to successfully adapt or acclimate to new environments. As such, they are critical for the growth and
survival of ectothermic animals living in marine environments with variable temperatures (Mosser et al.
1986; Hightower and Renfro 1988; Iwama et al. 1998).
The temperature range to which a fish species can adapt
is dependent upon adaptive cellular functions and stress
responses. For example, the adaptable temperature
ranges from 20 to 32°C for the experimental laboratory fish platyfish Xiphophorus maculatus and
zebrafish Danio rerio, and cultured cells of these species are also maintained in a similar temperature range
(Yamashita et al. 2004). Stress proteins, which are a
evolutionally well-conserved family of proteins, are
induced in response to a variety of stress conditions
and metabolic insults. Most stress proteins perform
essential biological roles as molecular chaperones that
facilitate the synthesis and folding of proteins and participate in protein assembly, secretion, trafficking, and
protein degradation (Pelham 1982; Lindquist 1986;
Lindquist and Craig 1988; Morimoto et al. 1990;
Georgopoulos and Welch 1993; Wu 1995; Hartl 1996;
© 2010 TERRAPUB, Tokyo. All rights reserved.
doi:10.5047/absm.2010.00304.0111
Received on February 2, 2010
Accepted on August 17, 2010
Online published on
December 29, 2010
Keywords
• activin
• development
• HSP70
• HSC70
• heat-shock transcription factor HSF
• molecular chaperone
• rainbow trout
• Nodal
• stress protein TGF-β
• transgenic zebrafish
Hartl and Hayer-Hartl 2002). When cells are subjected
to rapid changes in their environment, induced stress
proteins play a central role in the maintenance of cellular homeostasis. A response to sudden adverse environmental changes is referred to as the heat-shock or
stress response and is accompanied by a rapid increase
in the synthesis of stress proteins. Given the importance of stress proteins in thermal adaptation at the
cellular level, we focused on the expression, regulation, and protective functions of the heat-shock protein 70 (HSP70) protein family under normal and
stressed conditions in a variety of fish species.
2. HSP70 in fish
HSP70 is widely distributed group of HSPs found in
numerous organisms from bacteria to mammals, and
its expression is markedly induced in response to environmental stresses, such as heat shock, UV and γirradiation, and chemical exposure (Pelham 1982;
Lindquist 1986; Lindquist and Craig 1988; Morimoto
et al. 1990; Georgopoulos and Welch 1993; Wu 1995).
Transgenic and mutant zebrafish strains have been used
as model animals for stress research (Fig. 1) (Yamashita
1999; Yamashita et al. 2003; Yamashita and Hojo
2004). HSP70 is thought to have a molecular chaper-
M. Yamashita et al. / Aqua-BioSci. Monogr. 4: 111–141, 2010
112
B: western blot
A: immunostaining
Control
HSP70
Heat shocked
Fig. 1. Expression of HSP70 in the heat-shocked zebrafish embryo. Embryos maintained at 28.5°C were transferred to 37°C
for 1 h. The embryos were fixed with 10% formalin in PBS, and HSP70 was stained with a specific monoclonal antibody
(Embiotech Laboratories, Tokyo, Japan) vs. heat-inducible HSP70 raised against the C-terminal region of zebrafish HSP70-a
by detection of signals with BM Blue POD substrate (Roche Japan, Tokyo, Japan). (A) Whole mount immunostaining showed
HSP70 expressed in tissue specific manners. (B) Western blotting.
one function such that it transiently binds to nascent
polypeptides and unfolded proteins, thereby preventing intramolecular and intermolecular interactions that
can result in misfolding or aggregation of these
substrate proteins (Welch and Feramisco 1985) (Fig.
2). Many studies have examined gain- or loss-offunctions to elucidate the biological roles of HSP70 as
a molecular chaperone in animal cells in vitro and in
vivo. The chaperone functions of HSP70 appear to be
closely related to stress tolerance in animal cells, and
overexpression of HSP70 enhances anti-apoptotic activity against cellular stress (Feder et al. 1996; Kim et
al. 1997; Kondo et al. 1997; Ravagnan et al. 1997; Li
et al. 2000; Mosser et al. 2000a, b). The heatinducible gene expression and transcriptional regulation of the hsp70 gene has also been characterized in
ectothermic animals, such as rainbow trout and
zebrafish (Yamashita et al. 2004; Ojima et al. 2005a).
Two distinct isoforms of HSP70 cDNA have been
identified from both rainbow trout and zebrafish
(Yamashita et al. 2004; Ojima et al. 2005a). The amino
acid sequences of fish HSP70 are highly homologous
to heat-inducible-type HSP70 proteins in other vertebrates, such as human HSP70-1 and HSP70B′ (Fig. 3)
(Voellmy et al. 1985), with a very high identity (75–
80%), indicating that fish HSP70 proteins belong to
the heat-inducible HSP70 family. Molecular phylogeny
studies on all HSP70 proteins that have been identified in fish reveal two clusters, namely, “fish HSP701” or “fish HSP70-2” (Fig. 4), suggesting that these
diverged during vertebrate evolution (Yabu et al. unpublished). The two distinct HSP70 isoforms identified in rainbow trout, zebrafish, and platyfish are
thought to have subsequently evolved in fish.
2-1. HSP70 and other HSPs in rainbow trout
Stress responses have been well characterized in rainbow trout and its cultured cells. Currie and Tufts (1997)
observed that erythrocytes synthesized HSP70 both
constitutively and in response to an increase in temperature. Airaksinen et al. (1998) examined the effects
of heat stress (from 18 to 26°C) and low oxygen tension (1% O2 = 1 kPa) on protein synthesis in primary
cultures of hepatocytes, gill epithelial cells, and RTG2 cells of rainbow trout. All of these cells displayed
elevated levels of 67-, 69-, and 92-kDa proteins,
whereas a 104-kDa protein was induced only in RTG2 cells. Hypoxia induced a cell-type-specific response,
increasing the synthesis of 36-, 39-, and 51-kDa pro-
doi:10.5047/absm.2010.00304.0111 © 2010 TERRAPUB, Tokyo. All rights reserved.
M. Yamashita et al. / Aqua-BioSci. Monogr. 4: 111–141, 2010
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HSC70, HSP70
mtHSP70
Mitochondria
GRP78
Fig. 2. Model of molecular chaperone functions of the HSP70 family. HSP70 and HSC70 function as intracellular chaperones
for other proteins, regulating protein-protein interactions, such as protein folding, establishment of protein disassembly and
rearrangement, prevention of protein aggregation, transport of target proteins into the intracellular compartments, and translocation of proteins across membranes (Welch and Feramisco 1985). MtHSP70 and GRP78 are localized to the mitochondria
and the endoplasmic reticulum, respectively (Yamashita et al. 2004).
teins in the gill epithelial cells. When juvenile trout
reared in freshwater were transferred from freshwater
at 13.5°C to freshwater at 25.5°C, held for there for 2
h, returned to freshwater at 13.5°C for 12 h, and then
transferred to 32 ppt seawater at 13.5°C, the level of
branchial HSP70 increased approximately tenfold in
the heat-shocked fish relative to the control. Such a
mild temperature shock had only modest effects on the
ability of rainbow trout to resist osmotic stress during
fresh- to seawater transfer (Niu et al. 2008). HSP70
expression differs among rainbow trout clonal lines,
suggesting the genetic control of differences in HSP70
expression (Heredia-Middleton et al. 2008). Trout cells
have also been used in bioassays of heavy metal exposure in which the expression of HSP70 and
metallothioneins was the indicator of exposure
(Kothary and Candido 1982; Misra et al. 1989). In these
studies, HSP70 accumulated in juvenile trout tissues,
including the gill and liver, in response to exposure to
metal (Cd2+, Cu2+, Pb2+, Zn2+)-contaminated water and
diet. However, HSP70 levels in juvenile rainbow trout
did not increase significantly when the dissected tis-
sues were exposed individually to environmentally relevant Cd2+ or Cr2+ levels.
HSP70a and HSP70b have 98.1% identity in their
deduced amino acid sequences (Ojima et al. 2005a, b).
Southern blot analysis indicated that the two HSP70s
are encoded by distinct genes in the trout genome, and
northern blot analysis showed that heat stress of RTG2 cells resulted in each of HSP70a and HSP70b expressing two mRNA species of different sizes (Ojima
et al. 2005a). The induction levels of total HSP70b
mRNAs were observed to be consistently higher than
those of their HSP70a counterparts during heat stress,
although the expression profiles of the two genes were
similar to one another in temperature-shift and timecourse experiments. Moreover, a mRNA species with
a larger molecular size was expressed only under severe heat stress (i.e., not less than 28°C) irrespective
of HSP70a and HSP70b. Ojima et al. (2005b) isolated
multiple HSPs, including HSP90 β a, HSP90 β b,
glucose-regulated protein (GRP)78, HSP70a, heatshock cognate (HSC)70a, HSC70b, CCT8, HSP47, and
DnaJ homolog, from RTG-2 cells. Quantitative reverse
doi:10.5047/absm.2010.00304.0111 © 2010 TERRAPUB, Tokyo. All rights reserved.
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M. Yamashita et al. / Aqua-BioSci. Monogr. 4: 111–141, 2010
Fig. 3. Amino acid sequences of deduced proteins encoded in the heat-inducible hsp70 genes identified in the zebrafish
genome. Comparisons of the predicted amino acid sequences. Residues identical to the amino acid in the zebrafish HSP70-a
sequence are indicated by dots. Amino acids that are present in zebrafish HSP70-a, but not in other HSP70s, are marked by
dashes. The zebrafish cDNA sequence for HSP70-a has been deposited in the DDBJ database with the accession number
AB062116. The genes encoding HSP70-b and HSP70-ctg9500 are present on zebrafish chromosome 16 and 8, respectively
(Yamashita et al. 2004).
transcription (RT)-PCR analyses showed that the
mRNA accumulation levels of HSP70a, HSP70b,
HSC70a, HSC70b, and HSP47 were significantly elevated after heat shock, with those of two HSP70s,
HSP70a, and HSP70b, showing the greatest increase.
HSC70b showed a greater increase than HSC70a.
Ojima et al. (2005b) cloned two splice variants of
HSPB1 from the rainbow trout and found that the Cterminus of the deduced proteins had a polyglutamic
acid (polyE) stretch not found in other vertebrate
HSPB1s. These two splice variants, HSPB1_tv1 and
HSPB1_tv2, were identified in fish exposed to a continuous heat shock, with the mRNA level of
HSPB1_tv1 increasing in response to this stress while
that of HSPB1_tv2 decreased. Northern blot and RTPCR analyses showed that under normal physiological conditions, HSPB1_tv1 mRNA is predominantly
expressed in muscle tissues, although it is present in
all organs. In contrast, HSPB1_tv2 mRNA is selectively
expressed in muscle tissues, particularly in the heart.
Distinctive features of rainbow trout HSPB1, such as
having two splice variants and a polyE stretch, may
contribute to the function of the protein under the typical low-temperature habitat of cold-water fish.
2-2. Zebrafish HSP70
We isolated cDNA clones encoding heat-inducible
HSP70 from a cDNA library of 1-day-old zebrafish
embryos heat-shocked at 37°C for 1 h. The complete
cDNA sequence of a heat-inducible hsp70 in zebrafish
embryos has 77–79% homology to heat-inducible
hsp70 genes in platyfish, rainbow trout, and humans.
The zebrafish hsp70 gene exhibited heat-induced expression, as measured by Northern blotting and in situ
hybridization analyses, following a temperature shift
from 28.5 to 37°C. Krone et al. (1997) and Sueltmann
et al. (2000) reported a partial nucleotide sequence of
the 5′-flanking region of the zebrafish hsp70 and a
1,977-bp open reading frame (ORF), respectively. Nei-
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M. Yamashita et al. / Aqua-BioSci. Monogr. 4: 111–141, 2010
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sea bass HSC70
zebrafish HSC70-1
893
platyfish HSC70
yellowtail HSC70-2
933
999
Xenopus HSC70-2
Xenopus HSC70-1
291 986
HSC70
rat HSC70
853
1000 bovine HSC70
866
353
human HSC70
catfish HSC70
686
trout HSC70
745
yellowtail HSC70-1
chicken HSP70
469 rat HST70
1000
mouse HSP70.2
mammalian HST70
622
1000
human HSP70A2
449
bovine HSP70.3
1000
Chinook salmon HSP70
trout HSP70
950
1000
zebrafish HSP70-b
zebrafish HSP70-a
fish HSP70-2
322
Takifugu HSP70 (#482)
789
yellowtail
HSP70
931
Tilapia HSP70
601
884
560
medaka HSP70
752
platyfish HSP70-2
zebrafish HSP70 (#9500)
627
flounder HSP70
fish HSP70-1
616
1000
platyfish HSP70-1
395
Takifugu HSP70 (#1502)
Xenopus HSP70
human HSP70HOM
1000
rat HSP70.3
1000
mouse HSC70t
mouse HSP70.1
1000
942
rat HSP70.2
mammalian
999
rat HSP70.1
MHC-linked HSP70
1000
pig HSP70.1
bovine HSP70.2
975
890 human HSP70.2
1000 human HSP70.1
human HSP70B' mammalian HSP70B’
1000
pig HSP70B'
fly HSP70 (87A7)
ascidia HSP70
903
1000
0.02
Fig. 4. Molecular phylogenic tree of the HSP70 family members. Amino acid sequences of the vertebrate HSP70s were
compared by the neighbor-joining method with the CLUSTAL W program (version 1.83). Bootstrap confidence values for the
sequence groupings are indicated in the tree (n = 1000). The scale indicates the evolutionary distance of one amino acid
substitution per site. Sequence database accession numbers in GenBank or genomic contig scaffold numbers in the zebrafish
and Takifugu Ensenbl Genome Servers are indicated in parentheses.
ther of the clones were full-length cDNA, and mismatches in nucleotide sequences occurred between
them. The sequence reported by Sueltmann et al. (2000)
carries the same ORF as our isolated sequence up to
the point of heterogeneity, P-632, where a deletion of
a G at base 2557 occurs, after which the predicted
amino acid sequence diverges until a stop codon after
residue P-632; this sequence also lacks the C-terminal
conserved acidic amino acid sequence “EEVD.” Since
no apparent homology existed in the 3′-noncoding sequences, the sequence variations are probably due to a
distinct gene product. Thus, the zebrafish genome ap-
pears to contain another hsp70 gene, whose structure
and expression profile are different, as described in
Subsection 2-3.
Amino acid sequence homology analysis revealed
that the zebrafish HSP70 is a member of the heatinducible HSP70 family in vertebrates (Fig. 3) and that
it shares extensive homology with the heat-inducible
HSP70s from other fish species, such as rainbow trout
and platyfish (Fig. 3) (Yamashita et al. 2004). On the
other hand, two distinct mammalian groups have been
identified, namely, a Major Histocompatibility Complex (MHC)-linked group consisting of human HSP70-
doi:10.5047/absm.2010.00304.0111 © 2010 TERRAPUB, Tokyo. All rights reserved.
M. Yamashita et al. / Aqua-BioSci. Monogr. 4: 111–141, 2010
116
(B) human
human MHC-linked Hsp70 (Chr. 6)
Hsp70HOM Hsp70-2
Hsp70-1
Testis-specific Heat-inducible
Heat-inducible
(A) zebrafish
zebrafish hsp70-ctg9500 (Chr.8)
Constitutive
human Hsp70B’ (Chr. 1)
Heat-inducible
zebrafish hsp70 (Chr.16)
hsp70-a hsp70-b
Heat-inducible
human Hst70 (Chr. 14)
Constitutive
Testis-specific
Fig. 5. Gene structure of the hsp70 genes in zebrafish and human genomes. (A) The zebrafish genome contains three copies of
the hsp70 genes, and HSP70-a was the only heat-inducible member of the HSP70 family. The gene encoding HSP70-b is
tandemly linked to the gene encoding HSP70-a on the zebrafish chromosome 16, and the gene encoding HSP70-ctg9500 is
present on zebrafish chromosome 8 (Yamashita et al. 2004). Arrows indicate the heat-inducibility of these genes measured by
RT-PCR (Yabu et al. unpublished). (B) The human genome contains five copies of the hsp70 and its related genes; hsp70-1,
hsp70-2, and hsp70B′ are heat-inducible, whereas Hsp70HOM and Hst70 are testis-specific.
1 and HSP70-2 and mouse HSP70.1 (Hunt and
Morimoto 1985; Wu et al. 1985; Milner and Campbell
1990) and a group containing the human and pig
HSP70B′ (Voellmy et al. 1985; Gunther and Walter
1994). Thus, the heat-inducible HSP70s may have diverged into several distinct groups during vertebrate
evolution.
2-3. Gene structure of the hsp70 gene in zebrafish
Zebrafish and the tiger pufferfish Takifugu rubripes,
whose draft genomic sequences have been well documented, each have several copies of hsp70 genes (Figs.
5, 6). In zebrafish, a BLAST search of the Ensenbl
Zebrafish Genome Server localized the hsp70 genes
to two distinct loci on chromosomes 8 and 16 (Sanger
Institute, UK; Yamashita et al. 2004), with two
tandemly linked hsp70 genes, i.e., hsp70-a and -b, on
chromosome 16 (Fig. 5). The hsp70-a gene could be
induced in the cultured zebrafish cells and embryos
under heat-shock conditions (Fig. 6), whereas the
hsp70-b gene was weakly expressed constitutively,
based on RT-PCR results, suggesting that it may be a
pseudogene. In addition, an intron-less hsp70-like gene
on chromosome 8 showed constitutive expression in
zebrafish embryos under heat-shock conditions, indicating that this may also be a pseudogene. Thus, the
hsp70-a gene is a heat-inducible HSP70 isoform among
the HSP70 family proteins in the zebrafish genome.
2-4. Genomic analysis of fish hsp70 genes
Postlethwait et al. (1998) compared the zebrafish
genome organization with that of humans and found
that large chromosome segments have been conserved
for the 430 million years since the divergence of the
two lineages. These authors suggested that two largescale gene duplication episodes—possibly wholegenome duplication events—occurred prior to the divergence of the fish and mammal lineages. Analysis
of the zebrafish hox gene clusters revealed that the
zebrafish has two copies of human chromosome segments, suggesting that an additional genome duplication occurred in the zebrafish genome (Amores et al.
1998). A relationship between the divergence of hsp70
genes and a chromosomal duplication event was characterized by comparisons with zebrafish chromosomes
and mammalian orthologs. The genetic map of the
hsp70 genes in the zebrafish genome revealed a striking conservation of synteny with a region of human
and mouse chromosomes (Yamashita et al. 2004). The
almost orthologous genes in zebrafish chromosome 8,
which contains the hsp70 gene, were well conserved
on human chromosome 1 and mouse chromosome 3
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M. Yamashita et al. / Aqua-BioSci. Monogr. 4: 111–141, 2010
HSE
1st exon
1st intron
117
2nd exon
ORF
Heat shock promoter
Constitutive promoter
Fig. 6. The nucleotide sequence of the 5′-upstream region of the zebrafish hsp70-a gene. The transciption start site was
determined by primer extension. The nucleotide sequences in exons are underlined. The zebrafish hsp70 gene contains only
one intron in the 5′-noncoding region. The translation start ATG codon is in italics. The heat-inducible expression of the
zebrafish hsp70 gene is suggested to be regulated by a dual promoter system, i.e., a heat-shock promoter containing HSE
upstream of the transcription start site and another constitutive promoter in the first intron (Yabu et al. unpublished).
(Table 1). This finding indicates that zebrafish hsp70
(Chr. 8) is orthologous to human Hsp70B′, which localized to human chromosome 1q23.1. Therefore, the
fish hsp70-1 group, containing platyfish hsp70-1,
zebrafish hsp70 (Chr. 8), and Takifugu hsp70 (ctg1502),
is orthologous to the mammalian Hsp70B′. In contrast,
the orthologous genes of zebrafish hsp70-a on chromosome 16 could not be identified on human and
mouse chromosomes. The mammalian orthologous
genes corresponding to the fish genes on zebrafish
chromosome 16 were widely distributed on human
chromosomes 3, 12, 16, and 19 and mouse chromosomes 5, 6, 7, 8, 9, and 11 (Table 1). Thus, the mammalian ortholog of the fish hsp70-2 group appears not
to exist in the mammalian genome. The fish hsp70-2
group may have arisen by an ancient chromosomal
duplication event from the fish hsp70-1 group.
2-5. Stress-inducible HSC70 isoforms
In an earlier study, we established a cell line derived
from the tail fin of the yellowtail Seriola
quinqueradiata, a marine teleost fish, and isolated
cytosolic HSP70 and two HSC70 isoforms (Yabu et
al. unpublished). We observed that the heat-shock regulation of these two hsc70 genes is quite distinct, and
named them hsc70-1, which is constitutively expressed,
and hsc70-2, which is only induced by heat shock (Yabu
et al. unpublished). In addition, HSP70/HSC70 was
translocated into the lysosomes, and chaperonemediated autophagy (CMA) assisted by HSP70/HSC70
was induced in the heat-shocked cells (Yabu et al. unpublished). The hsp70 gene possesses a cis-acting heatshock element (HSE) responsive to heat-induced transcriptional activation (Amin et al. 1988; Xiao and Lis
1988; Wu 1995). Thus, under stress conditions, both
hsp70 and hsc70-2 genes may be regulated by a heatshock factor via a HSE in the promoter. In rainbow
trout cells, the promoter region of the hsc70 gene possesses potential HSE sequences, but its stressinducibility is unclear (Zafarullah et al. 1992). HSE
sequences have also been found in the promoter regions of mammalian hsc70 genes that lack significant
heat-shock inducibility (Sorger and Pelham 1988). Two
doi:10.5047/absm.2010.00304.0111 © 2010 TERRAPUB, Tokyo. All rights reserved.
Chr. 16
Huntingtin interacting protein B (9.0 Mb)
Centaurin a2 (9.1 Mb)
Mitogen-activated protein kinase 3 (9.2 Mb)
FOS-related antigen (9.2 Mb)
Calcium ATPase (9.3 Mb)
HSP70-a and -b (9.3 Mb)
Kinesin family member C3 (9.4 Mb)
Non-muscle myosin heavy chain 14 (9.8 Mb)
60S ribosomal protein L18 (10.0 Mb)
CD27L receptor (10.0 Mb)
TNF-R1 (10.1 Mb)
HSP70 (Chr. 8) (17.3 Mb)
Adenosylhomocysteinase 2 (17.3 Mb)
CCAAT-binding transcription factor subunit B (17.3 Mb)
Neugrin (17.3 Mb)
Sortilin precursor (17.3 Mb)
Mitsugumin 29 (17.4 Mb)
Unknown (17.4 Mb)
Chr. 8
Monocarboxylate transporter 5 (MCT 5) (17.0 Mb)
RNA-binding protein 15 (17.0 Mb)
Homeobox protein aristaless-like 3 (17.0 Mb)
Sodium- and chloride-dependent neurotransmittertransporter NTT4 (17.2 Mb)
Potassium voltage-gated channel subfamily cmember 4 (17.2 Mb)
Zebrafish genes in the chromosome containing the HSP70 gene (localization)
NM_012271 (Chr. 3, 47.0 Mb)
NM_024857 (Chr. 17, 29.3 Mb)
MAPK3 (Chr. 16, 30.2 Mb)
NM_024816 (Chr. 16, 29.0 Mb)
ATP2A1 (Chr. 16, 28.9 Mb)
not found
KIFC3 (Chr. 16, 57.6 Mb)
NM_024729 (Chr. 19, 55.4 Mb)
RPL18 (Chr. 19, 53.8 Mb)
TNFRSF7 (Chr. 12, 6.4 Mb)
TNFRSF1A (Chr. 12, 6.3 Mb)
SLC16A4 (Chr. 1, 110.2 Mb)
RBM15 (Chr. 1, 110.2 Mb)
ALX3 (Chr. 1, 109.9 Mb)
NTT4 (Chr. 1, 110.0 Mb)
KCNC4 (Chr. 1, 110.1 Mb),
KCNC3 (Chr. 1, 110.5 Mb)
HSPS6 (Chr. 1, 158.8 Mb)
AHCYL1 (Chr. 1, 109.8 Mb)
NFYA (Chr. 6, 41.1 Mb)
NM_016645 (Chr. 15, 88.5 Mb)
SORT1 (Chr. 1, 109.2 Mb)
ENSG00000143028 (Chr. 1, 109.3 Mb)
NM_153340 (Chr. 1, 109.3 Mb)
Q8BL12 (Chr. 9, 113.2 Mb)
NM_172133 (Chr. 11, 79.7 Mb)
MK10 (Chr. 5, 102.1 Mb)
NM_030566 (Chr. 7, 118.6 Mb)
NM_007504 (Chr. 7, 118.6 Mb)
not found
KFC3 (Chr. 8, 96.4 Mb)
NM_028021 (Chr. 7, 37.6 Mb)
RL18 (Chr. 6, 130.3 Mb)
TNR7 (Chr. 6, 127.2 Mb)
TR1A (Chr. 6, 127.3 Mb)
NM_146136 (Chr. 3, 110.4 Mb)
Q7TT82 (Chr. 3, 110.5 Mb)
ALX3 (Chr. 3, 110.7 Mb)
NM_172271 (Chr. 3, 110.6 Mb)
NM_145922 (Chr. 3, 110.6 Mb),
CIK3 (Chr. 3, 110.2 Mb)
not found
NM_145542 (Chr. 3, 110.8 Mb)
CBFB (Chr. 17, 47.8 Mb)
NM_031375 (Chr. 7, 72.6 Mb)
NM_019972 (Chr. 3, 111.4 Mb)
MG29 (Chr. 3, 111.4 Mb)
NM_175183 (Chr. 3, 111.3 Mb)
Human homolog (chromosomal localization) Mouse homolog (chromosomal localization)
Table 1. Zebrafish genes neighboring the HSP70 genes.
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hsc70 genes, hsc70-1 and hsc70-2, were expressed in
the hepatopancreas and muscle of carp under unstressed
conditions in a characteristic tissue-specific manner
(Ali et al. 2003). These were insensitive to or only
weakly induced by the stressors, with two exceptions:
cadmium treatment and cold-shock-induced hsc70-1 in
the liver and enhanced induction of hsc70-2 in the
muscle (Ali et al. 2003). Thus, the fish genome possesses two genetically distinct hsc70 genes, the hsc701 and hsc70-2 groups.
When we compared gene synteny of hsc70 genes in
the zebrafish genome, the hsc70-1 gene was found to
be orthologous to the human hsc70 gene, showing similar gene synteny, whereas the hsc70-2 gene was different from the human orthologs (Yabu et al. unpublished). This finding suggests that two distinct hsc70
genes showing different expression patterns under heat
stress and other stress conditions might be present in
the fish genomes. In zebrafish, the hsc70-1 gene is
constitutively expressed in cultured cells as well as
expressed in a tissue-specific manner, particularly in
the brain and yolk sac (Graser et al. 1996; Yamashita
et al. 2003), whereas the hsc70-2 gene is not expressed
in the cultured cells, suggesting that the hsc70-2 gene
may be silent. On the other hand, members of the
hsc70-2 group, such as platyfish HSC70 (Yamashita
et al. 2004), medaka HSC70 isoforms
[NP_001098384], Seriola HSC70-2, and carp HSC702 (Ali et al. 2003), are expressed in the respective fish
tissues. Therefore, both hsc70-1 and hsc70-2 genes may
act as molecular chaperones in different tissues and
cell types under normal and/or stressed conditions. The
vertebrate HSP70/HSC70 proteins, including the heatinducible HSP70, testis-specific protein, the MHClinked HSP70, and the constitutively expressed HSC70,
are postulated to have evolved from four distinct groups
by gene duplication and translocation in the vertebrate
genome as potential adaptations to various environmental stresses (Yamashita et al. 2004). Among these proteins, teleost fish possess two copies of HSC70, which
was duplicated during fish evolution. The cytosolic
HSP70/HSC70 may be responsible for CMA under both
normal and stressed conditions (Yabu et al.
unpubilished).
3. Heat-shock transcription factor in fish
Heat-inducible transcriptional regulation is mediated
by the heat-shock transcription factor (HSF) that binds
to HSEs found upstream of all stress protein genes
(Morimoto et al. 1990; Wu 1995). The human, chicken,
and tomato genomes contain multiple distinct hsf genes
(Scharf et al. 1990; Rabindran et al. 1991; Sarge et al.
1991; Schuetz et al. 1991; Nakai and Morimoto 1993),
whereas the genomes of yeast, Drosophila, and Xenopus each contain only a single hsf gene (Sorger and
Pelham 1988; Wiederrecht et al. 1988; Mercier et al.
119
1997). In mammals and chickens, HSF1 and HSF3 were
identified as rapidly activated, stress-responsive factors (Rabindran et al. 1991; Sarge et al. 1991; Nakai
and Morimoto 1993; Nakai et al. 1995). HSF2 responds
to erythrocyte differentiation and preimplantation embryonic development (Hensold et al. 1990; Schuetz et
al. 1991; Sistonen et al. 1992), and HSF4 acts as both
an activator and a repressor of tissue-specific heatshock genes through alternative splicing (Nakai et al.
1997; Tanabe et al. 1999). The HSF proteins have
highly conserved amino-terminal regions that contain
the DNA-binding domain for HSE as well as hydrophobic heptad repeats that form coiled-coil structures.
In mammals and Drosophila, the inactive monomeric
form of HSF is converted to a trimer in response to
heat and other stresses, possibly through a switch from
intramolecular to intermolecular coiled-coil interactions (Westwood et al. 1991; Rabindran et al. 1993;
Zuo et al. 1994). HSF binds to the promoter region
and activates transcription through a potent heatactivation domain in its C-terminus, which is negatively regulated in the absence of stress (Green et al.
1995; Shi et al. 1995; Zuo et al. 1995; Newton et al.
1996; Wisniewski et al. 1996; Kline and Morimoto
1997).
3-1. Zebrafish HSF
The nucleotide sequence of the isolated zebrafish
HSF cDNA was predicted to encode a full-length HSF
of 497 amino acids (accession No. BAB72171) (Fig.
7). Comparison of this amino acid sequence to other
previously characterized members of the HSF family
revealed that the zebrafish HSF belongs to the HSF1
group consisting of human, mouse, and chicken HSF1.
These proteins regulate the heat-inducible
transactivation of the stress genes. The amino acid sequence of zebrafish HSF is 57, 55, and 57% identical
to human, mouse, and chicken HSF1, respectively. The
DNA-binding domain of the zebrafish HSF is well conserved with previously reported HSF1s (Fig. 7). The
amino acid sequence of the DNA-binding domain of
the zebrafish HSF1 is 92, 92, and 94% identical to the
corresponding region in human, mouse, and chicken
HSF1, respectively. Other conserved motifs found in
the zebrafish HSF include the hydrophobic repeat that
functions as the oligomerization motif, which is 70%
identical to the corresponding region of human HSF1.
The carboxyl-terminal heptad repeat, which plays a role
in intramolecular negative regulation of DNA-binding
activity in HSF1, is also conserved in zebrafish HSF.
The zebrafish HSF shares extensive homology with
the rainbow trout HSF (Fig. 7). Components found in
other vertebrate HSF1s, such as the DNA-binding domain, predicted nuclear localization signal, hydrophobic heptad repeat (HR)-A/B and HR-C, are conserved
in the zebrafish HSF. Thus, HSF transactivation of
doi:10.5047/absm.2010.00304.0111 © 2010 TERRAPUB, Tokyo. All rights reserved.
120
M. Yamashita et al. / Aqua-BioSci. Monogr. 4: 111–141, 2010
DNA-BINDING DOMAIN
HYDROPHOBIC HEPTAD REPEAT A/B
HEAT-ACTIVATION DOMAIN
HYDROPHOBIC HEPTAD REPEAT C
Fig. 7. Comparisons of the predicted amino acid sequence of zebrafish HSF with those of human HSF1, mouse HSF1, chicken
HSF1, and rainbow trout HSF1a proteins. Residues identical to the amino acid in the zebrafish HSF (accession number
AB062117) are indicated by dots. Amino acids that are present in zebrafish HSF, but not in other HSFs, are marked by dashes.
The regions boxed with a solid line or broken line indicate the predicted DNA binding domains and hydrophobic heptad
repeats, respectively. The potent heat-activation domain boxed in red is suggested to regulate temperature ranges for HSF
activation and conformational changes.
stress genes is a common phenomenon in fish and
mammals. However, the question as to whether other
types of HSF, such as HSF2, HSF3, and HSF4, which
have been reported in humans and chickens, are also
present in fish, remains to be resolved.
A recent study by Rabergh et al. (2000) reported the
nucleotide sequences of two splice variants of the
zebrafish hsf gene. There are several differences between sequence reported by us and that reported by
these authors. In particular, we noted the following
amino acid substitutions: P-252 to S, P-254 to S,
SALTPP (342–347) to FRPDSA, L-430 to Val,
IVLPDPL (446–452) to SFSPIPF, and A-478 to T.
These substitutions are due to nucleotide heterogeneity and frameshifts caused by nucleotide insertions and
deletions. The C-terminal amino acid sequence, KLS
(496–498), was well conserved in the known HSFs,
while in the sequence of Rabergh et al. (2000) it was
replaced with SRTRIGDPCFKLKKESKR by an insertion of the unknown nucleotide sequence after the nucleotide at position 1631, which is suggested to be due
to a splicing variant containing introns. Although these
structural differences in HSF may affect DNAbinding and transactivation properties, as previously
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M. Yamashita et al. / Aqua-BioSci. Monogr. 4: 111–141, 2010
A: hsp70 mRNA
121
B: hsf mRNA
Fig. 8. Northern blot analysis of hsp70 and hsf mRNAs from zebrafish embryos. Total RNA was isolated from embryos
incubated at 28.5°C and heat-shocked embryos that had undergone a 1-h temperature shift to 37°C, and then subjected to
Northern analysis. (A) hsp70 mRNA, (B) hsf mRNA. The hsf gene was expressed as a primary transcript of 6 kb and a mature
form of 2 kb after the gastrula stage, which coincided with the expression pattern of the hsf gene. Embryonic staging was
according to Kimmel et al. (1995).
pointed out in studies on Drosophila (Wu 1995), their
functional properties have not been examined (Rabergh
et al. 2000).
3-2. Rainbow trout HSF1
Two distinct cDNA clones encoding HSFs have been
isolated from RTG-2 cells of rainbow trout and subsequently denoted HSF1a and HSF1b (Ojima and
Yamashita 2004). The predicted amino acid sequence
of HSF1a shows 86.4% identity to that of HSF1b. The
two proteins contained the general structural motifs of
HSF1, i.e., a DNA-binding domain, hydrophobic heptad repeats, and nuclear localization signals. Southern
blot analysis showed that each HSF1 is encoded by a
distinct gene. The two HSF1 mRNAs were coexpressed in unstressed rainbow trout RTG-2 cells as
well as in a variety of tissues. An electrophoretic mobility shift assay revealed that each in vitro translated
HSF1 binds to the HSE, and a chemical cross-linking
and immunoprecipitation analysis showed that HSF1a
and HSF1b form heterotrimers as well as homotrimers.
Based on these results, two distinct HSF1 isoforms that
can form heterotrimers are present in rainbow trout
cells, suggesting that a unique molecular mechanism
regulated by a combination of distinct HSF1 isoforms
underlies the stress response in rainbow trout. These
HSF1 isoforms may have diverged during the evolution of tetraploid fish.
4. Transcriptional control of fish hsp70
The expression of the hsp70 and hsf genes in
zebrafish embryos at six different life-cycle stages was
examined by Northern blot analysis (Fig. 8). hsp70
mRNA levels were found to be very low in control
embryos incubated at 28.5°C under non-stress conditions; however, these dramatically increased at each
stage following heat shock, with the exception of the
3-h embryos (blastula stage), in which no hsp70 expression was observed. hsf mRNA was not found in 3h embryos, but appeared as both 2-kb and 6-kb molecules in the embryos after 6 h and thereafter. In
zebrafish embryos, hsp70 mRNA was expressed after
the gastrula period under heat-shock conditions, as
identified by Northern blot analysis. The results of the
in situ hybridization experiment indicated that the
hsp70 gene was expressed in a stage- and tissuespecific manner. The hsp70 mRNA was localized in
the brain, eye, otic vesicle, and yolk sac under heat
stress conditions. Given that hsf mRNA was expressed
in the embryos after the gastrula period, the hsf gene
is thought to be induced by zygotic expression during
early development, which is similar to most other
housekeeping genes. Based on the observation that hsf
mRNA expression paralleled the heat-inducible expression of hsp70, hsf expression after the gastrula period
may accompany the heat induction of the zebrafish
hsp70 gene in a stage- and tissue-specific manner.
hsp70 expression patterns were examined in vivo by
whole-mount in situ hybridization (Fig. 9). During the
cleavage and blastula periods, no apparent expression
of hsp70 mRNA occurred even in heat-shocked 3-h
embryos. However, heat-shock induction of hsp70
mRNA was found in the embryos 6 h after fertilization. A temperature elevation from 28.5 to 37°C for 1
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M. Yamashita et al. / Aqua-BioSci. Monogr. 4: 111–141, 2010
122
B
A
C
D
Fig. 9. Heat-inducible expression of the hsp70 gene detected by in situ hybridization. The fish hsp70 gene showed stressinducible gene expression. In zebrafish embryos, hsp70 mRNA was expressed at the segmentation stage after the gastrula
period under heat-shock conditions. During the cleavage and blastula periods, no apparent expression of hsp70 mRNA was
seen, even in the heat-shocked 3-h embryos (B). However, heat-shock induction of hsp70 mRNA was found in the embryos 6
h after fertilization (D). A temperature shift from 28.5°C to 37°C for 1 h induced hsp70 mRNA expression throughout the
entire embryo during the gastrula and segmentation periods (B), (D). Conversely, control embryos cultured at 28.5°C showed
no apparent expression of hsp70 mRNA in any of the stages tested in this study (A), (C).
h induced hsp70 mRNA expression throughout the entire embryo during the gastrula and segmentation periods. Most notably, strong expression was observed
in the brain, notochord, and yolk sac of heat-shocked
1- to 2-day-old embryos. In the 2-day heat-shocked
embryos, hsp70 mRNA expression was elevated in the
forebrain, hindbrain, notochord, otic vesicle, yolk sac,
and skeletal muscle in the middle part of the body (Fig.
10). Conversely, control embryos cultured at 28.5°C
showed no apparent expression of hsp70 mRNA in any
of the stages tested in this study (Fig. 9).
The expression levels of hsf mRNA were also examined (Fig. 11). hsf mRNA was not observed in embryos
within 3 h of fertilization, as shown by the Northern
blot analysis (see Fig. 8), but it was present throughout all developmental stages of zebrafish embryos after the gastrula period (Fig. 11). In 2-day-old embryos,
hsf mRNA was highly expressed in the brain, notochord, otic vesicle, and yolk sac. Thus, the
spatiotemporal expression of zebrafish hsf mRNA accounts for the tissue-specific heat-inducibility of
HSP70 under stress conditions.
Fig. 10. Heat-inducible expression of the hsp70 gene detected by in situ hybridization. Most notably, strong expression of the hsp70 gene was observed in the brain, notochord,
and yolk sac of heat-shocked 1- to 2-day-old embryos. In
the case of the 2-day-old heat-shocked embryos, hsp70
mRNA expression was elevated in the forebrain, hindbrain,
notochord (nt), otic vesicle (ov), yolk sac (yc), and skeletal
muscle (m) in the middle part of the body. Thus, the hsp70
gene was expressed in stage- and tissue-specific manners.
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M. Yamashita et al. / Aqua-BioSci. Monogr. 4: 111–141, 2010
A
B
123
C
D
Fig. 11. Expression of hsf mRNA by in situ hybridization. The hsf gene was expressed after the gastrula period. The hsf gene
is induced by zygotic expression during early development, similar to most other housekeeping genes. hsf mRNA was not
observed in embryos within 3 h of fertilization (A) as shown by Northern blot analysis (see Fig. 8). After the gastrula period,
hsf mRNA was observed throughout the developmental stages of zebrafish embryos (B)–(D). In 2-day-old embryos, HSF
mRNA was highly expressed in the brain, notochord (nt), otic vesicle (ov), and yolk sac (yo) (D). Thus, the spatiotemporal
expression of zebrafish hsf mRNA accounts for the tissue-specific heat-inducibility of HSP70 under stress conditions.
4-1. Stress responses in embryos
hsp70 mRNA is expressed after the gastrula period
under heat-shock conditions, as reported in previous
studies (Bienz 1986). Since hsf mRNA is expressed in
embryos after the gastrula period, the hsf gene is probably induced by zygotic expression during early development in a manner similar to that of other housekeeping genes (Newport and Kirschner 1982). hsf
mRNA expression also paralleled the heat-inducible
expression of hsp70, which is mainly regulated by HSF.
Therefore, hsf expression after the gastrula period is
likely accompanied by heat induction of the hsp70 gene
in a stage-specific manner.
In mice, the MHC-linked heat-inducible Hsp70.1
gene exhibits complex transcriptional regulation in
response to serum factors, infection, development, and
stress (Milner and Campbell 1990; Morimoto et al.
1990). This gene is transcribed during the cleavage
period and also after gastrulation in response to cell
cycling, but not to stress induction (Bevilacqua and
Mangia 1993; Christians et al. 1997). The mouse and
human Hsp70 genes located in the MHC locus are expressed by transcription factors other than HSF1
(Morimoto et al. 1990; Wu 1995). Conversely, given
that the zebrafish hsp70 shows strict heat-inducible
expression, it appears to undergo a simple transcriptional regulation that may involve only HSF, a system
that is similar to that observed for the Drosophila and
human HSP70B′ (Craig et al. 1979; Voellmy et al.
1985; Morimoto et al. 1990; Gunther and Walter 1994).
The overexpression of HSF constructs lacking the
regulatory domain result in the expression of hsp70
and hsp47 in embryos, even under non-stress conditions. This finding indicates that the deleted regulatory domain plays an important role in repressing the
conversion of the inactive monomer to an active trimer.
The zebrafish HSF is considered to play a major role
in the stress-inducible expression of hsp70 and other
hsp genes in zebrafish embryos. HSF monomers associate with each other, resulting in the formation of
homotrimers in response to physiological stress and
ultimately activating target hsp genes by acquiring
DNA-binding ability (Wu 1995; Zhong et al. 1998).
Phosphorylation of serine and threonine residues in
HSF has also been reported to be involved in a signal
transduction system, including mitogen-activated protein kinase (MAPK) pathways (Knauf et al. 1996). A
number of authors have reported that hsf expression
regulates in vivo thermotolerance in Drosophila and
mice (Jedlicka et al. 1997; Xiao et al. 1999) and cultured mammalian cells (Mivechi et al. 1995; McMillan
et al. 1998). Thus, the spatiotemporal expression of
hsf in embryos reported here may determine the heatinducible expression of hsp genes during zebrafish
development, thus causing the embryos to be resistant
against environmental stresses, such as changes in temperature.
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M. Yamashita et al. / Aqua-BioSci. Monogr. 4: 111–141, 2010
124
1
2
3
4
hsp70-GFP
hsp70-GFP + HSF
hsp70-GFP + Del1
hsp70-GFP + Del2
5
hsp70-a
hsp47
hsc70-1
hsf
Fig. 12. Transactivation activities of zebrafish HSF. (A) Gene expression induced by overexpression of HSF. The cRNA
encoding zebrafish HSF (HSF, lane 2) and the HSF mutants, HSF-Del1 (Del1, lane 3) and HSF-Del2 (Del2, lane 4), was
introduced into zebrafish embryos at the one-cell stage. The embryos were then incubated at 28.5°C for 12 h. The control
embryos were maintained at 28.5°C (control, lane 1), and the heat-shocked embryos were incubated at 37°C from the 10 h
post-fertilization (hpf) to the 12 hpf (heat shock, lane 5). In the 12-h embryos, the mRNA expression levels of hsp70-a, hsp47,
hsc70-1, and hsf were examined by RT-PCR. (B) The cRNA encoding zebrafish HSF or that of the truncated mutants, HSFDel1 (Del1) and HSF-Del2 (Del2), was microinjected into zebrafish embryos at the one-cell stage, along with the hsp70-GFP
gene construct. The induction of GFP expression under the control of the hsp70 promoter was observed in 12-h embryos by
fluorescence microscopy.
4-2. Transactivation by the cloned zebrafish HSF
We investigated the transcriptional activities of the
zebrafish hsf to confirm that the cloned hsf regulates
the heat-inducible expression of zebrafish hsp70. The
hsf cDNAs that encode the protein with a fused Histag at the C-terminal and its truncated forms were
subcloned downstream of a T7 promoter and their
cRNAs subsequently synthesized using in vitro transcription.
Expression vectors encoding HSF or mutant HSFs
were introduced into zebrafish embryos, and the induced levels of the stress genes were assayed by RTPCR to examine whether the zebrafish HSF functions
as a transcriptional activator (Fig. 12). Embryos containing the deletion mutants HSF-Del1 and HSF-Del2,
in which the heat-activation domain between HR-A/B
and HR-C, respectively, of HSF is deleted, expressed
high levels of mRNA encoding hsp70 and hsp47 in a
pattern similar to that seen with the heat-induced expression of hsp70 and hsp47. Conversely, the fulllength HSF clone did not induce the expression of
hsp70 and hsp47 when introduced into embryos. As a
control experiment, the zebrafish hsc70 gene was constitutively expressed in every embryo tested in this
study. These findings indicate that the zebrafish HSF
without the heat-activation domain can transactivate
hsp70 and hsp47 under non-stress conditions and that
the full-length hsf is negatively regulated under nonstress conditions. When the cRNA encoding zebrafish
HSF or the truncated mutants, HSF-Del1 (Del1) and
HSF-Del2 (Del2), was microinjected into zebrafish
embryos at the one-cell stage, along with the hsp70GFP gene construct, the induction of GFP expression
under the control of the hsp70 promoter was observed
in 12-h embryos by fluorescence microscopy (Fig. 12).
The introduction of the truncated HSF (e.g., Del1 and
Del2) induced green fluorescence, indicating that the
truncated sequence negatively regulated the transcription of the hsp70 promoter. In contrast, the introduction of original HSF did not induce green fluorescent
protein (GFP) expression under the control of the hsp70
promoter.
5. Characterization of the HSP70 family members
in vertebrates
The HSP70 family consists of several members, some
of which are heat inducible and others that are constitutively expressed (Morimoto et al. 1990). Both HSP70
doi:10.5047/absm.2010.00304.0111 © 2010 TERRAPUB, Tokyo. All rights reserved.
M. Yamashita et al. / Aqua-BioSci. Monogr. 4: 111–141, 2010
and HSC70 are cytosolic members of the HSP70 family. HSP70 is markedly induced under a variety of
stresses, including heat shock, UV irradiation, and
treatment with heavy metals or arsenite, while HSC70
is expressed under normal growth conditions
(Morimoto et al. 1990). GRP78 is located in the endoplasmic reticulum and induced under the inhibitory
condition of glycosylation (Morimoto et al. 1990). A
testis-specific HSP70-related protein found in the mammalian testis has been shown to be involved in spermatogenesis (Allen et al. 1988; Matsumoto and
Fujimoto 1990). These HSP70 family members have
evolutionarily diverged, and they have been classified
by phylogenetic analysis into four distinct clusters corresponding to their intracellular localization, i.e., the
cytoplasm, endoplasmic reticulum, mitochondria, or
chloroplasts (Morimoto et al. 1990; Boorstein et al.
1994).
In addition, multiple HSP70 isoforms have been
found in various animal cell types by two-dimensional
polyacrylamide gel electrophoresis (2D-PAGE) analysis (Welch and Feramisco 1985; Allen et al. 1988;
Matsumoto and Fujimoto 1990; Gutierrez and
Guerriero 1995). These isoforms are assumed to be due
to the presence of the multiple Hsp70 gene copies that
have been identified in humans (Wu et al. 1985;
Gunther and Walter 1994), mice (Hunt et al. 1993;
Gunther and Walter 1994), and Drosophila (Craig et
al. 1979). Alternatively, posttranslational modifications, such as methylation, ribosylation, and phosphorylation, of HSP70 might account for isoform diversity.
5-1. The HSP70 family members in fish cells
Yamashita et al. (2004) characterized the expression
of HSP70 family proteins in the platyfish fibroblast
cell line EHS and isolated three distinct cDNA clones
that encoded two isoforms of HSP70 and HSC70. A
phylogenetic analysis of the heat-inducible members
of the HSP70 family in vertebrates revealed four distinct groups. This study demonstrates that a shift in
the incubation temperature of platyfish EHS cells from
28 to 37°C apparently induces a set of stress proteins,
including HSP28, HSP70, HSP90, GRP78, and GRP94,
in a manner similar to that seen in mammalian and
Drosophila cells (Morimoto et al. 1990). Several additional spots of 40, 35, 32, and 25 kDa, indicating
small HSPs induced by the stress treatments, have also
been found, but these have never been characterized
in fish cells (Yamashita et al. 2004).
The anti-HSP70 antibody shows a broad specificity
against HSC70 and HSP70 in animal cells (BRM-22;
Sigma-Aldrich, St. Louis, MO), and a specific rabbit
antibody has been raised against the N-terminal sequences derived from the platyfish cDNA clones. Both
of these results indicate the stress-inducible expres-
125
MHC-linked HSP70
mammalian MHC-linked
HSP70
MHC-linked testis specific
HSP70 related protein
Mammalian testis-specific HST70
Mammalian HSP70B’
Bird HSP70
Amphibian HSP70
Fish HSP70-1
Fish HSP70
Fish HSP70-2
Fig. 13. The model of divergence of the HSP70 family mem-
bers during vertebrate evolution. The hsp70 genes may have
diverged during vertebrate evolution several times. The duplication of the hsp70 genes may have occurred during fish
evolution (open circle) or mammalian evolution (close circle).
sion of the HSP70-1 and HSP70-2 isoforms. The antibody against the constitutively expressed HSC70 also
cross-reacted with the two immunologically crossreactive proteins by differential phosphorylation of a
single gene product (Yamashita et al. 2004). Phosphorylated isoforms of HSP70 have been also found in bovine cells (Leustek et al. 1992). The kinase and the
locations of the phosphorylated amino acid residues
in HSC70 remain unknown and should be characterized.
5-2. Phylogenetic analysis of fish HSP70
Phylogenetic analysis has demonstrated that the
eukaryotic HSP70 can be classified into four distinct
clusters, such as HSP70/HSC70, GRP78, mitochondrial
HSP70, and plastid HSP70, according to their intracellular localization in the cytoplasm, endoplasmic reticulum, mitochondria, or chloroplasts, respectively
(Boorstein et al. 1994). In addition, the Hsp70 genes
in humans and mice constitute a multigene family (Wu
et al. 1985; Hunt et al. 1993; Gunther and Walter 1994).
The HSP70 family protein genes are distinguished by
at least three different expression patterns: strictly heatinducible Hsp70 (e.g., human Hsp70B; Voellmy et al.
1985), cell cycle-dependent, heat-inducible Hsp70
(e.g., human Hsp70-1 and Hsp70-2; Hunt and
Morimoto 1985; Milner and Campbell 1990), and constitutively expressed, less stress-dependent Hsp70
genes (e.g., Hsc70; Ali et al. 1996; Hsp70Hom; Milner
and Campbell 1990). The Hsp70 genes are distributed
on at least five different human chromosomes (Gunther
and Walter 1994; Yamashita et al. 2004). The amino
acid homology analysis showed that the heatinducible members of the vertebrate HSP70 family can
be classified by adding new members of fish HSP70
family into six clusters, tentatively named “fish
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M. Yamashita et al. / Aqua-BioSci. Monogr. 4: 111–141, 2010
126
HSP70,” “mammalian testis-specific HST70,” “mammalian HSP70B′,” “mammalian MHC-linked HSP70,”
“bird HSP70,” and “amphibian HSP70”, respectively
(Fig. 13).
Platyfish HSP70-1 and HSP70-2, rainbow trout
HSP70, and zebrafish HSP70 belong to the “fish
HSP70” cluster. Since the expression of these fish
HSP70 is inducible at high levels in fish cells, the
HSP70 proteins in this group are considered to be the
major heat-inducible forms. According to the
phylogenetic analysis in Fig. 4, fish HSP70 can be classified into two genetically distinct groups, “fish
HSP70-1” and “fish HSP70-2.” The presence of two
HSP70 isoforms belonging to these groups suggests
the duplication of the heat-inducible hsp70 gene during evolutionary divergence in fish.
5-3. Mammalian HST70
The second cluster “mammalian HST70” contains the
testis-specific, constitutively expressed mouse hsp70.2
as well as its human and rat orthologs. To date, no
member of this cluster has ever been identified in lower
vertebrates (Yamashita et al. 2004). Both the mouse
homolog hsp70.2 gene and the other testis-specific
hsp70-related hsc70t gene regulate testicular formation and spermatogenesis (Allen et al. 1988;
Matsumoto and Fujimoto 1990). These mammalian
Hst70 genes have an intron in the 5′-flanking sequence
similar to the other heat-inducible fish hsp70s, but they
are expressed in a testis-specific manner. Thus, mammalian HST70s may have diverged structurally and
functionally from the heat-inducible hsp70 by the replacement of a testis-specific promoter (Gunther and
Walter 1994). These findings suggest that mammalian
Hst70s are important in testicular function specific to
homeotherms, but not in the lower vertebrates.
5-4. Mammalian MHC-linked HSP70
Two heat-inducible Hsp70s encoded in the mammalian MHC class III locus are classified into the “mammalian MHC-linked HSP70” cluster. In the human genome, three copies of Hsp70-related genes are
tandemly localized in the MHC system locus on chromosome 6p21 (Gunther and Walter 1994). The first and
second of these genes encode heat-inducible Hsp70,
whereas the third gene in the sequence encodes the
constitutively expressed testis-specific HSP70-related
protein in the opposite orientation to the neighboring
Hsp70 genes (Hunt and Morimoto 1985; Milner and
Campbell 1990; Gunther and Walter 1994). This gene
organization is also conserved in the mouse and rat
(Gunther and Walter 1994). Conversely, the MHC system locus in the zebrafish genome does not contain
the homologous gene to the mammalian MHC-linked
Hsp70, suggesting that three copies of the MHC-linked
Hsp70 genes may have been duplicated from an ancestral MHC-linked Hsp70 gene during mammalian
evolution. In addition, the third gene (i.e., human
Hsp70Hom (Milner and Campbell 1990), mouse hsc70t
(Matsumoto and Fujimoto 1990), and rat hsp70.3
(Walter et al. 1994)] of the three tandemly linked hsp70
genes in the mammalian MHC locus is testis-specific.
Their promoter regions lack HSEs and display testisspecific expression in mammalian spermatogenesis
(Allen et al. 1988; Matsumoto and Fujimoto 1990).
5-5. Mammalian HSP70B′′
The syntenic analysis of the zebrafish genome revealed that the fish HSP70-1 group containing
zebrafish hsp70 (Chr. 8) is orthologous to the human
Hsp70B′ on human chromosome 1q23.1. The pig genome contains a similar Hsp70B′ gene, whereas the
mouse and rat genomes have lost this gene during mammalian divergence. Comparison of the promoter functions of the two distinct human Hsp70 gene promoters
(Morimoto et al. 1990) revealed that the gene encoding Hsp70B′ exhibits simple transcriptional regulation,
which is similar to that found in yeast and Drosophila.
Conversely, the MHC-linked heat-inducible Hsp70
genes exhibit complex transcriptional regulation and
respond to diverse cellular signals, such as serum factors, viral activation, developmental regulation, and
stress induction (Hunt and Morimoto 1985; Milner and
Campbell 1990). Both human promoters have conserved HSEs that can bind the heat-shock transcription factor (Hunt and Morimoto 1985; Wu et al. 1985;
Milner and Campbell 1990; Morimoto et al. 1990;
Gunther and Walter 1994).
6. Stress response in transgenic zebrafish models
Transgenic zebrafish models expressing stress proteins, HSF, and reporter genes under the control of the
heat-shock promoter have been generated and used for
stress research in fish with the aim to observe the stress
response and temperature regulation in vivo (Yamashita
et al. 2003). The minimum hsp70 promoter containing
a single HSE and showing high heat-inducible transcriptional activity was used to visualize the stress response occurring in intact embryos using the GFP gene
expression system. In addition, transgenic zebrafish
overexpressing HSF showed enhanced stress tolerance.
The expression of stress proteins induced by HSF activation may determine the stress response and stress
tolerance in vivo.
6-1. Transgenic zebrafish as a biosensor for
stresses
The gene expression of hsp70 is regulated by a heatshock promoter. Its transcription is activated by
doi:10.5047/absm.2010.00304.0111 © 2010 TERRAPUB, Tokyo. All rights reserved.
M. Yamashita et al. / Aqua-BioSci. Monogr. 4: 111–141, 2010
changes in body temperature as well as a variety of
other stresses. For this reason, HSP70 is considered to
be a useful marker protein of the stress conditions of
animals. The molecular basis for this induction has
been attributed to its regulatory regions, which effectively direct the production of heterologous proteins
under stress conditions. The transgenic nematode,
which possesses lacZ driven by the hsp16-1 gene promoter, shows different tissue-specific effects following heat-shock and chemical stresses (Stringham et al.
1992). Heat shock was observed to affect the entire
body, whereas chemical stressors affected only specific
organs, such as the pharynx and nerve ring. The
transgenic mouse with an introduced hsp68-lacZ gene
construct shows notable heat-shock responses of the
transgene in the amnion, epidermis, heart, and neural
tissues in a tissue-specific manner (Kothary et al.
1989). In addition, a transgenic mouse with a human
growth hormone under the control of the human Hsp70
promoter was found to release growth hormone in the
plasma following intraperitoneal injection with a toxic
compound (Sacco et al. 1997). The results of these studies support the concept that such transgenic animals
make useful models for examining the conditions causing stress responses and cellular damage. The organic
basis for this induction has been identified to the regulatory regions, which effectively direct the production
of heterologous proteins under stress conditions.
Induction of the hsp70 gene in a stress-inducible
manner is essentially regulated by a transcriptional
mechanism. Previous attempts to introduce a mouse
and human Hsp70 promoter into fish (Liu et al. 1990;
Bayer and Campos-Ortega 1992) suggest that mammalian promoters have only low transcription activity.
Since the HSE contains three mismatched nucleotides,
in comparison to the trout, and the consensus HSE sequences, we used the minimum hsp70 promoter containing the consensus HSE sequence and generated a
consensus sequence by base substitution of the three
mismatched nucleotides and then fused the construct
to the bacterial lacZ (Fig. 14). Transgenic zebrafish
were produced by microinjecting a one-cell stage embryo with the circular form of the plasmid DNA of the
lacZ construct (Yamashita 1999). DNA from the tail
fins of the transgenic individuals of the F0 generation
was then screened by PCR. The integrated transgene
was detected in 32 F0 individuals among the 120 F0
fish screened, and germ line transmission was found
in 15 F0 individuals. Among the transgenic fish, an F1
transgenic founder, tentatively named the P7 strain,
showed high β-galactosidase activity only under stress
conditions. Fish embryos derived from a cross between
an F1 female and a wild type were fixed and stained
using X-gal before and after being exposed to heat
shock. Following a temperature increase from 28.5 to
37°C for 6 h, high levels of lacZ expression were observed in every tissue of the embryo. In comparison,
127
control
As
HS
Fig. 14. Expression of the β -galactosidase gene under the
control of the hsp70 promoter. Each 72-h-stage transgenic
zebrafish of the F1 generation was fixed and stained with Xgal (Yamashita 1999). The control transgenic fish (control)
was cultured at 28.5°C for 72 h after fertilization. The DNA
construct of the hsp70 promoter-lacZ gene was microinjected
into the cytoplasm of the one-cell-stage embryo. The injected
embryos were cultured for 5 days in sterilized tap water in
an incubator at 28.5°C, and juvenile fish were then cultivated to adult fish. The transgenic F1 generation was treated
with 100 µM sodium arsenite for the last 8 h (As) or heatshocked in warm water at 37°C for the last 6 h (HS). The
tissue-specific expression of the transgene was induced in
the lens and yolk extension by sodium arsenite.
treatment with a chemical stressor, sodium arsenite,
affected lacZ expression in a tissue-specific manner.
Specifically, the eyes and intestinal organs showed high
β-galactosidase activity. These results show that heatshock stress affects all tissues in the body, whereas the
chemical stressor affects only the specific target tissues and cells. A state of acute arsenite toxicity was
reached within 24 h in the transgenic zebrafish, at an
estimated 560 µ M; in contrast, the chemical stressorspecific transgene expression in the eye and the intestine was observed at 5 µM of sodium arsenite (Fig.
15). This experiment demonstrates that an approach
using transgenic fish carrying the hsp70-lacZ gene
construct provides an easy procedure for in vivo toxicity monitoring in aquatic environments. Therefore, the
hsp70 promoter used here is considered to be one of
the most active and useful promoters in fish
transgenesis.
In the case of transgenic mice carrying a growth hormone gene driven by the human Hsp70 promoter,
transgene expression, as determined by the plasma
growth hormone level, occurred in mice injected with
2.5 mg/kg of sodium arsenite as well as in the culture
cells treated with 10–50 µ M sodium arsenite or other
inorganic compounds (Sacco et al. 1997). The promoter
region of the Hsp70 gene regulates heat-inducible expression through HSE, corresponding to inverted repeats of GAA and TTC. The heat induction of the
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128
M. Yamashita et al. / Aqua-BioSci. Monogr. 4: 111–141, 2010
the expression pattern of the hsc70 gene in transgenic
zebrafish carrying the bacterial β-galactosidase lacZ
reporter gene under the control of the trout hsc70 promoter. The hsc70 genes have also been isolated and
characterized from rainbow trout and zebrafish
(Zafarullah et al. 1992; Graser et al. 1996). hsc70 was
expressed in a tissue-specific manner in the brain,
retina, lens, spinal cord, and yolk sac in zebrafish embryos during the hatching period. Thus, this promoter
is available for examining overexpression of a specific
gene under non-stress conditions for the generation of
transgenic fish.
6-2. Enhanced stress tolerance via the expression
of active HSF
Fig. 15. Dose response of the β -galactosidase gene expres-
sion under the control of the hsp70 promoter to sodium arsenite. The transgenic embryos showing β -galactosidase
activity in the eyes were counted, and 50% of the fish positive for β -galactosidase gene expression could be determined
at 5 µM sodium arsenite (filled triangles), whereas lethality
(50% lethal dose, LD50) following a 24-h arsenite treatment
was observed at 560 µM (filled circles). This finding indicates that transgene expression is a more sensitive marker
than lethality by approximately 100-fold.
transgene was found in every tissue in the body, with
especially strong expression occurring in the yolk sac
and eye, whereas chemical induction by arsenite was
restricted to the eye and part of the intestine. Given
that no sequences other than the HSE were found in
the minimum promoter used for the expression system
in the present study, the tissue-specific expression by
arsenite might have been due to tissue-specific uptake
and/or unknown chemical responsive transactivation
mechanisms mediated by HSF.
Yamashita et al. (2003) observed that the stressinducible expression of the transgene was easily detected in situ by induced fluorescence in GFP
transgenic fish, enabling analysis of stress responses
in fish in their aquatic environment. The specific GFP
expression by the transgene has also been observed in
living cells and tissues in embryos (Fig. 16). Such an
inducible gene expression system can be used as a
bioassay tool for measuring responses to aquatic stress
stimuli, such as hyper- and hypoosmolarity, temperature, environmental contaminants, radiation, and UVirradiation.
In addition to its application as a stress-inducible
promoter, the hsc70 promoter can be used for fish
transgenesis. HSC70 is a constitutive expressing member of the HSP70 family in animal cells. This protein
is considered to play a chaperone function, supporting
protein folding and the transmembrane under normal
growth conditions. Yamashita et al. (2003) examined
Environmental stresses, such as heat shock, UVirradiation, infection, and toxic chemicals, affect the
growth and physiological conditions of fish in aquatic
environments. Biotechnology offers the possibility to
modify and improve fish physiological properties.
However, an important challenge remains—that of
identifying the genes that can improve stress tolerance.
Heat-inducible transcriptional regulation is known
to be mediated by HSF, which binds to HSE present
upstream of every type of stress protein gene. The
known HSF proteins share high sequence conservation
in an N-terminal region comprising the DNA-binding
domain, which recognizes HSE in the heat-shock gene,
and hydrophobic heptad repeats that form coiled-coil
structures (Wu 1995; Xiao et al. 1999). Inactive HSF
is monomeric under normal physiological conditions
and converted to a trimer in response to heat and other
stresses, possibly through a switch from intramolecular to intermolecular coiled-coil interactions (Fig. 17)
(Wu 1995; Xiao et al. 1999). Once bound to the promoter, HSF activates transcription through a potent
heat-activation domain in the C-terminus, which is
negatively regulated in the absence of stress (Jedlicka
et al. 1997; Nakai et al. 2000). We isolated a cDNA
for a zebrafish HSF1 homolog that was predicted to
encode a full-length HSF of 497 amino acids. When
the cRNA encoding a mutant HSF lacking the possible
heat activation domain was transiently introduced into
zebrafish embryos, the overexpression resulted in the
expression of hsp70 and hsp47 in embryos even under
non-stress conditions (Fig. 12). Thus, the expression
of HSF mutants lacking a heat-activation domain can
induce various types of the stress protein genes regulated by the heat-shock promoter and HSF even under
non-stress conditions.
Nakai et al. (2000) observed that, in transgenic mice
in which an active HSF mutant was expressed, many
HSP genes were overexpressed under normal growth
conditions. These transgenic mice showed growth arrest of testis tissue, but the relationship between the
overexpression of HSF and stress tolerance has never
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M. Yamashita et al. / Aqua-BioSci. Monogr. 4: 111–141, 2010
129
D
Fig. 16. Transgenic zebrafish expressing green fluorescent protein (GFP) under the control of the heat-shock promoter. Heat
shock (A) and γ-irradiation (B) induced the expression of the hsp70-GFP transgene in some surface cells in the head and yolk
sac in the living transgenic embryos in comparison with no apparent GFP expression in the control embryo (C). Photo shows
fluorescent view of the transgenic embryos at 36 hpf. Panel D indicates the gene construct of the GFP gene under the control
of the rainbow trout heat shock promoter (Yamashita 1999).
been characterized in mice in vivo. Drosophila mutants
showing tolerance against heat stress have also been
established (Tanabe et al. 1998). These mutants possess point mutations in the DNA-binding domain in
HSF and express high levels of HSPs under non-stress
conditions. The mutants showed apparent tolerance
against heat-shock stress during developmental stages.
In contrast, an in vitro study using mouse and chicken
cultured cells reported that disruption of the HSF gene
reduced stress tolerance. Additionally, in an in vivo
assay, a knockout mouse showed multiple phenotypes,
such as defects of the chorioallantoic placenta and prenatal lethality, growth retardation, female infertility,
elimination of the classical heat-shock response, and
exaggerated tumor necrosis factor alpha production
(Xiao et al. 1999). These reports suggest that HSF1
regulates critical physiological events during
extraembryonic development and under pathological
conditions both in vivo and in vitro. However, the relationship between the expression of HSPs regulated
by HSF and the stress tolerance remains to be elucidated in vertebrates in vivo.
To establish zebrafish cell lines expressing HSF
mutants, we generated two kinds of expression
plasmids containing the zebrafish HSF cDNA lacking
the heat-activation domain under the control of the
CMV promoter (Fig. 17). The two transgenic lines,
Del1 and Del2, carrying the plasmids, pCMV-HSFdel1
and pCMV-HSFdel2, respectively, were generated by
microinjection of plasmids into zebrafish embryos. The
transgene product mutant HSF with a His-tag sequence
in the C-terminus end of the protein was detected by
Western blotting in all transgenic fish lines of homozygous F2 progenies (Fig. 18). We selected the two
lines (i.e., Del1 and Del2) that exhibited the highest
expression levels of mutant HSF in embryos for further evaluation.
The overexpression of the active form of the HSF
mutant was confirmed by Western blotting and a gelmobility shift assay. The integration of the transgene
was confirmed by PCR amplification. The transgenic
fish was found to express both HSFs by Western blotting (Fig. 18). Both transgenic lines, Del1 and Del2,
showed higher levels of the transgene product than the
wild-type fish.
To provide an understanding of the relationship between cellular HSF levels and stress tolerance in vivo,
we examined the survival rates of transgenic zebrafish
that overexpressed HSF under stressed conditions using UV-irradiation as a model. When transgenic fish
embryos were exposed to UV irradiation at 12 hpf (hour
post-fertilization), the HSF-expressing transgenic fish
had higher survival rates in a dose-dependent manner
than wild-type fish (Fig. 19). These findings show that
HSF overexpression determines stress tolerance.
The transgenic expression of the active form of HSF
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M. Yamashita et al. / Aqua-BioSci. Monogr. 4: 111–141, 2010
130
heat-activation
domain
1: Del1
2: Del1
3: Del2
4: Del2
5: wild-type
Del1
Del2
DNA-binding
domain
Fig. 18. Transgene expression in zebrafish. Induced expres-
DNA-binding
domain
sion of HSP70/HSC70 was assayed in the embryos
overexpressing HSF mutants Del1 and Del2. Transgenic
embryos (lanes 1, 2: Del1 individual; lanes 3, 4: Del2 individual; lane 5: wild-type embryo) at 48 hpf were used for
Western blotting with anti-His-Tag (Roche) and anti-bovine
HSC70 (Sigma-Aldrich) antibodies.
Del1
Del2
Fig. 17. Activation of HSF. (A) Model of HSF activation
under stress conditions as described in Yamashita et al.
(2003). (B) The HSF constructs lacking a heat-activation
domain used to obtain stress-resistant transgenic lines. The
nucleotide sequences of the gene constructs have been deposited to the DDBJ/EMBL/GenBank DNA Database
(AB062118-AB062120).
in zebrafish embryos effectively enhanced stress tolerance in vivo. Given that the transgenic embryos expressed stress proteins, the enhanced stress tolerance
in the fish embryo was likely due to overexpression of
stress genes, such as hsp70, hsp47, and hsp28, which
are upregulated by the active form of HSF.
Conversely, the disruption of the hsf1 gene in mouse
cells and the hsf3 gene in chicken cells leads to a loss
of the heat-shock response (Tanabe et al. 1998). In
addition to previous studies that support HSF being
closely related to the stress response in vivo, our data
clearly demonstrate that the active HSF determines
stress tolerance in vertebrates at the whole organism
level in vivo, indicating that the transition of HSF from
the inactive form to the active trimer is critical for the
improvement of stress tolerance. Given that the amino
acid substitution of the DNA-binding domain and heatactivation domain in HSF caused the activation of
HSF1 by a conformation change even under normal
non-stress conditions, such a sequence substitution in
the hsf gene may modulate stress tolerance in fish and
other animals in vivo.
Engineered stress tolerance in fish has not been previously reported. The transgenesis of the active HSF
form could be extended to other fish and animals as
well as cultured cells, thereby providing tolerance to
broad ranges of environmental stresses, such as heat,
UV-irradiation, hyperosmolarity, heavy metals, toxic
chemicals, and infection. Moreover, other stressresponsive transcription factors, such as heavy metal-
Fig. 19. Survival of transgenic embryos following UV irradiation. The embryos were maintained at 28.5°C after UVirradiation at 60 mJ/cm2 and 12 hpf. Survival rate was assayed for wild-type zebrafish (open circle) and transgenic
strains with Del1 mutant (filled triangle) or Del2 mutant
(filled circle). Each value represents the mean of three independent experiments. Error bars represent one standard deviation (n = 30).
responsive transcription regulator (MTF) and interferon
regulatory transcription factor (IRF), could be used
such that gene transfer of their active forms is mutated
into transactivation domains of transgenic fish and
animals to improve other types of stress tolerance
(Radtke et al. 1995).
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M. Yamashita et al. / Aqua-BioSci. Monogr. 4: 111–141, 2010
6-3. Overexpression of HSP70
Numerous studies have examined gain- or loss-offunction to elucidate the biological roles of HSP70 as
a molecular chaperone in animal cells in vitro and in
vivo. Overexpression of HSP70 in mammalian cultured
cells has been shown to enhance anti-apoptotic activity against cellular stress (Kim et al. 1997; Kondo et
al. 1997; Ravagnan et al. 1997; Li et al. 2000; Mosser
et al. 2000a, b). HSP70 blocks apoptosis by binding
apoptosis protease activating factor-1 (Apaf-1), thereby
preventing constitution of the apoptosome (Apaf-1/
cytochrome C/caspase-9 activation complex) (Beere et
al. 2000; Saleh et al. 2000; Xanthoudakis and
Nicholson 2001). In contrast, TCR/CD3- and Fasmediated apoptosis is enhanced by HSP70
overexpression in vitro (Liossis et al. 1997). In
transgenic mice and rats, HSP70 overexpression in the
heart was found to improve survival after the animals
were subjected to ischemic heart injury (Marber et al.
1995; Hutter et al. 1996). Extra copies of the HSP70
gene resulted in increased inducible thermotolerance
in transgenic Drosophila larvae (Feder et al. 1996).
Thus, the chaperone functions of HSP70 appear to
closely regulate thermotolerance in animal cells. However, whether the enhanced HSP70 levels improve
stress tolerance in vertebrates at a whole-organism level
in vivo has never been elucidated.
During the early stages of development, heatinducible HSP70 can be induced by heat shock as well
as other kinds of stress after the gastrula stage
(Bensaude et al. 1983; Christians et al. 1995; Krone et
al. 1997). In comparison, HSC70, a constitutively expressed member of the HSP70 family, is present as a
maternal protein in oocytes and early embryos before
gastrulation (Bensaude et al. 1983). Although the
HSP70 and HSC70 proteins found in embryos are
thought to be associated with target proteins as molecular chaperones under both normal and stressed
developmental conditions, the physiological role of
HSP70/HSC70 proteins in vivo remains unclear.
To investigate the biological significance of the enhanced HSP70 levels in early development, we generated and characterized transgenic zebrafish lines that
overexpressed HSP70 (Yamashita and Hojo 2004).
Unexpectedly, constitutive expression of HSP70 resulted in extensive apoptosis in the zebrafish embryos.
We report here several unique features of the transgenic
lines that are apparently associated with stress tolerance and the proapoptotic mechanism regulated by the
chaperone function of HSP70.
Yamashita and Hojo (2004) generated HSP70overexpressing transgenic zebrafish that stably expressed platyfish HSP70 cDNA driven by the rainbow
trout hsc70 promoter. This gene construct was linked
to the GFP gene under the control of the CMV promoter. The use of the GFP reporter gene as a marker
131
enabled easy selection of transgenic embryos. The plasmid DNA containing the hsc70 promoter-HSP70 cDNA
linked to the CMV promoter-GFP construct was introduced by microinjection into single-cell zebrafish embryos, and transgenic fish were obtained in the F1 generation. Since the transgenic embryos displayed brightgreen GFP fluorescence, F1 embryos stably expressing the transgene were easily selected. Two distinct
transgenic lines were generated, tentatively named SG1
and SG2, respectively. The integration of the transgene
was confirmed by PCR amplification. Western blotting revealed that the transgenic fish expressed both
HSP70 and GFP. The transgenic lines SG1 and SG2
showed different levels of transgene HSP70 product,
with the SG1 line having a higher expression of the
introduced HSP70 protein than the SG2 line.
Transgene expression was verified by western blotting and RT-PCR of DNA extracted from the tail fin of
F1 transgenic fish (Yamashita and Hojo 2004). Detection with anti-bovine HSP70 monoclonal antibody
showed that the transgenic lines expressed approximately 2.4-fold higher levels of HSP70 than wild-type
fish. Because of its broad specificity against HSP70/
HSC70 proteins, this antibody cross-reacted to both
the transgene product platyfish HSP70 protein and the
endogenous zebrafish HSP70/HSC70 proteins in the
wild-type fish. RT-PCR analysis also revealed the specific expression of the transgene product, namely,
platyfish HSP70 mRNA.
During early development, transgenic embryos
showed extensive apoptosis that induced abnormal
morphogenesis (Yamashita and Hojo 2004), and by 5
days postfertilization, 12–25% of the embryos were
dead.
Fluorescent
staining
of
terminal
deoxynucleotidyltransferase-mediated dUTP nick-end
labeling (TUNEL) revealed the presence of
fluorescent-labeled apoptotic cells throughout the embryos, particularly in the eyes, brain, and spinal cord.
In addition, when HSP70 cRNA was microinjected into
single-cell-stage zebrafish embryos, the HSP70 cRNA
also induced extensive apoptosis and abnormal morphogenesis. The degeneration of the whole embryo and
notochord was indicated by HSP70 overexpression.
When the caspase inhibitor benzyloxycarbonyl-ValAla-Asp-fluoromethylketone (Z-VAD-fmk) was comicroinjected with HSP70 cRNA into zebrafish embryos, the inhibitor prevented the apoptosis induced
by HSP70 overexpression. However, when an HSP70
mutant lacking the C-terminal peptide binding domain
was microinjected, no induction of abnormal morphogenesis was observed. These findings indicate that
HSP70 overexpression induced a caspase-mediated
apoptotic pathway by a chaperone function through the
C-terminal peptide-binding domain.
The spatial and tissue-specific nature of the observed
apoptosis was most likely relevant to the expression
pattern of the transgene under the control of the HSC70
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M. Yamashita et al. / Aqua-BioSci. Monogr. 4: 111–141, 2010
132
Wild-type
HSP70+
Anti-HSP70
Wild-type
HSP70+
Anti-P-Smad2
Fig. 20. Induction of Smad2 phosphorylation in the transgenic zebrafish embryos at 9 hpf by whole-mount staining with
antibodies vs. phosphorylated Smad2 and HSP70. The embryos overexpressing platyfish HSP70 under the control of the
HSC70 promoter (Yamashita and Hojo 2004) were fixed with 10% formalin in PBS, and phosphorylated Smad2 and HSP70/
HSC70 were stained with anti-phosphorylated Smad2 rabbit monoclonal antibody (Cell Signaling) and anti-bovine HSC70
(Sigma-Aldrich) by detection of signals with BM Blue POD substrate (Roche Japan).
promoter (Yamashita and Hojo 2004). These results
suggest that HSP70 is able to induce extensive
apoptosis in zebrafish embryos. Although the surviving embryos developed into adults, the transgenic fish
showed defects in the jaw, had small eyes, and lacked
a tail fin (Yamashita and Hojo 2004). The transgenic
adult fish overexpressing HSP70 showed abnormal
morphogenetic development and a defect of the upper
jaw.
We have successfully generated transgenic zebrafish
that overexpress HSP70 (Yamashita and Hojo 2004).
However, the fish show extensive apoptosis and abnormal morphogenesis in early development. Feder et
al. (1996) reported that the expression of a higher
number of HSP70 transgene copies resulted in en-
hanced heat tolerance in transgenic Drosophila,
whereas the protective role of HSP70 has been demonstrated in transgenic mice with heart injury (Marber
et al. 1995; Hutter et al. 1996). The transient
overexpression of HSP70 in cultured cells is considered to be protective against stress-inducible apoptosis
(Kim et al. 1997; Kondo et al. 1997; Ravagnan et al.
1997; Li et al. 2000; Mosser et al. 2000a, b). In contrast, our findings indicate that HSP70 overexpression
during zebrafish development brings about stress sensitivity. As HSP70 is believed to exhibit chaperone
functions under cellular stress conditions, the HSP70
overexpression in early embryos may regulate the expression pattern of various target proteins that bind to
HSP70. Also, bone morphogenetic protein-4 (BMP4)
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M. Yamashita et al. / Aqua-BioSci. Monogr. 4: 111–141, 2010
mRNA level has been found to be higher in transgenic
embryos than in wild-type embryos, suggesting that
the enhanced HSP70 levels regulate the BMP4mediated apoptotic signaling in early development. The
transgenic embryos overexpressing HSP70 showed
enhanced levels of phosphorylated Smad2 (Fig. 20).
Smad2 is phosphorylated by the activation of activinlike kinases, including BMP4 and Nodal receptors, via
ligand binding. This finding suggests that the receptor
may be activated by the overexpression of HSP70.
BMP4 is known to negatively regulate head and
somite formation (Graham et al. 1994; Furuta et al.
1997; Trousse et al. 2001). Given that high HSP70 levels were found to induce BMP4 signaling and repress
head formation in this study, we propose that in order
for morphogenesis to occur in early development,
HSP70 is required to manifest BMP4-mediated
apoptosis-inducing activity by regulating downstream
genes and/or serving as one component of multiple
inductive signals. As BMPs have been shown to regulate interdigital cell death in the avian embryo, the
undifferentiated distal mesodermal cells may undergo
chondrogenic differentiation or apoptosis depending on
whether they are incorporated into the future digital
rays or into the interdigital spaces (Merino et al. 1999).
Both chondrogenesis and apoptosis are induced by local BMPs. BMP pro-apoptotic activity is reported to
regulate the expression of members of the msx family
of closely related homeobox-containing genes and is
finally mediated by caspase activation (Merino et al.
1999). These findings indicate that HSP70 may modulate the ability of BMPs to induce apoptosis through
an upstream signaling factor present in the early embryos, such as members of the fibroblast growth factor (FGF) and Transforming growth factor beta (TGFβ) families.
7. Molecular chaperone function of HSP70 in
zebrafish embryos
7-1. Control of TGF-β receptor activation
Several gain- or loss-of-function studies have been
performed to elucidate the biological roles of HSP70
as a molecular chaperone in animal cells in vitro and
in vivo (Marber et al. 1995; Feder et al. 1996; Hutter
et al. 1996; Kondo et al. 1997; Liossis et al. 1997;
Ravagnan et al. 1997; Beere et al. 2000; Mosser et al.
2000a, b; Saleh et al. 2000; Xanthoudakis and
Nicholson 2001). We recently demonstrated that the
overexpression of HSP70 in zebrafish embryos induces
BMP4 expression and extensive apoptosis during early
development, which results in ventralized phenotypes
(Yamashita and Hojo 2004). Therefore, our current
focus is on the molecular chaperone functions of the
HSP70 family proteins in Activin/Nodal/TGF- β
signaling mechanisms responsible for vertebrate dors-
133
oventral formation.
Members of the TGF- β superfamily are involved in
many biological activities, including growth, differentiation, migration, cell survival, and adhesion in both
the diseased and normal states (Whitman 2001; Schier
2003). They are classified into two major groups:
Activin/Nodal/TGF-β and BMP/GDF. TGF-β ligand
binding induces the formation of a receptor complex
that consists of receptor type II and type I, both of
which are required for signal transduction (Luo and
Lodish 1996; Renucci et al. 1996; Lawler et al. 1997;
Garg et al. 1999; Nagaso et al. 1999). Both type II and
type I receptors contain serine/threonine kinase domains in their intracellular portions. Type II receptor
kinases are constitutively active and, upon ligand binding, hetero-tetrameric complexes, composed of two
molecules each of the type II and type I receptors, are
formed (Luo and Lodish 1996; Lawler et al. 1997). In
the tetrameric receptor complexes, type II receptor
kinases transphosphorylate type I receptors, which are
thereby activated and phosphorylate intracellular
substrates, i.e., transcription factor Smad proteins
(Derynck et al. 1998; Muèllera et al. 1999; Miyazawa
et al. 2002). To date, several cofactors, including extracellular proteins, Lefty proteins, Cerberus,
Tomoregulin-1, and the transmembrane protein Dpr2,
have been found to be associated with the receptors
and to inhibit Nodal signaling during early embryogenesis (Whitman 2001; Schier 2003; Zhang et al.
2004). However, intracellular cofactors have been
poorly characterized with respect to the receptors for
Nodal signaling. In this study, we characterized the
activation of Nodal receptors by HSP70/HSC70 with
the aim of investigating the molecular chaperone function and the target protein of HSP70/HSC70 in early
development.
Zebrafish hsc70 is expressed as a maternal protein,
and mRNA in the fertilized eggs and its zygotic gene
expression occur after gastrulation, especially in the
brain and yolk sac (Graser et al. 1996; Santacruz et al.
1997). When segmentation starts, hsc70 is expressed
in the dorsal trunk neural tube, the lateral plate mesoderm, and the tail bud. This expression pattern of hsc70
suggests a role in early mesoderm induction.
Since HSC70 deficiency reduces the expression of
the gsc and lim1 genes, genetic interactions likely occur between HSC70 and Nodal signaling. Activin and
Nodal have mesoderm-inducing abilities producing a
variety of mesoderm derivatives in a dosedependent manner (Whitman 2001; Schier 2003). In
zebrafish, the type I receptors consist of Activin
receptor-like kinase (ALK)2 and ALK4, while type II
receptors comprise ActRIIB (Luo and Lodish 1996;
Renucci et al. 1996; Lawler et al. 1997; Garg et al.
1999; Nagaso et al. 1999). Nodal signaling can be promoted by phosphorylation of the transcription factor
Smad2, which is mediated by Nodal/Activin/TGF-β
doi:10.5047/absm.2010.00304.0111 © 2010 TERRAPUB, Tokyo. All rights reserved.
M. Yamashita et al. / Aqua-BioSci. Monogr. 4: 111–141, 2010
134
(A) Dimerization of Type II receptor
(B) Formation of Ligand-receptor complex
Ligand
Type I
receptor
Type II
receptor
active Type I
receptor
Type II
receptor
P
P
P
HSC/HSP70
P
P
Smad2
P
P-Smad2
Transcription of
target genes
Fig. 21. Model for the formation and activation of Activin/Nodal/TGF- β and BMP receptors by HSP70/HSC70. HSP70/
HSC70 binds directly to the type II receptor kinases of the receptors and facilitates Nodal signaling.
receptors. The HSC70 deficiency caused by
microinjection of the Morpholino antisense oligo,
HSC-MO, which is a synthetic inhibitor against translation of the hsc70 gene, is accompanied by an apparent reduction in Smad2 phosphorylation at the shield
stage (Yamashita et al. unpublished).
The phosphorylation of Smad2 was observed to be
inhibited in embryos treated with the type I receptor
kinase inhibitor SB-431542 (Muèllera et al. 1999; Ho
et al. 2006). This inhibitor did not affect the expression of HSC70. In addition, Smad2 phosphorylation
was induced following the microinjection of HSP70
cRNA into zebrafish one-cell-stage zebrafish embryos
(Yamashita et al. unpublished). It can therefore be concluded that Smad2 phosphorylation by Activin-like
receptor kinase is regulated by the expression levels
of HSC70. The detection of phosphorylated Smad2 is
an important marker for Nodal signaling enhanced by
HSC70 in zebrafish embryos. To characterize the molecular mechanism of enhanced Nodal signaling by
HSC70, we examined the relationship between the type
I and type II receptors and stress proteins (Yamashita
et al. unpublished).
A constitutively active form of the type I serine/threonine kinase receptor Taram-A* type I receptor shows
kinase activity in the absence of any ligand-receptor
complex formation with the type II receptor (Renucci
et al. 1996). This reaction is not inhibited by HSCMO injection (Yamashita et al. unpublished). Therefore, Smad2 phosphorylation is mediated by the type I
receptor alone and is not regulated by HSC70.
During the activation process of tetrameric receptor
complexes, the ActRIIB kinase acts as an upstream
component of the type I receptor in the signaling pathways (Luo and Lodish 1996; Renucci et al. 1996;
Lawler et al. 1997; Garg et al. 1999; Nagaso et al.
1999). Overexpression of ActRIIB kinase following the
injection of cRNA into the zebrafish embryos induced
the phosphorylation of Smad2. In addition, coinjection of ActRIIB kinase cRNA and HSC-MO inhibited Smad2 phosphorylation (Yamashita et al. unpublished). Therefore, the activation of the type II
receptor in Nodal signaling is regulated by HSC70. The
expression patterns of the constitutive HSC70 and heatinducible hsp70 genes may affect the complex pattern
of Nodal expression during early embryogenesis. Since
HSP70 is induced and accumulated under stress conditions, these protein levels may affect the patterning
of morphogenesis mediated by Nodal and other TGFβ family ligands (Yamashita et al. unpublished).
In this study, we propose a novel molecular chaperone function of HSP70/HSC70 on the kinase activation of type II receptor, i.e., ActRIIB kinase (Fig. 21).
First, the serine residue around the catalytic center,
which is the primary autophosphorylation site for kinase activation of the type II receptor, is phosphorylated by an intermolecular mechanism assisted by
HSP70/HSC70. Second, autophosphorylation of other
serine and tyrosine residues is accompanied by
homodimerization of the type II receptor. Furthermore,
the type II receptor kinase phosphorylates the cytoplasmic domain of the type I receptor at serine and threonine, and the phosphorylation of both types of cytoplasmic domain contributes to the stability of the
heteromeric complex. Therefore, as a cytosolic molecular chaperone, the HSC70 protein plays essential roles
doi:10.5047/absm.2010.00304.0111 © 2010 TERRAPUB, Tokyo. All rights reserved.
M. Yamashita et al. / Aqua-BioSci. Monogr. 4: 111–141, 2010
135
Chaperone-mediated autophagy
HSP70/HSC70
substrate proteins
lysosome
macroautophagy
Fig. 22. Induction of autophagy. Two pathways, i.e., CMA and macroautophagy, are characterized in fish cells (Yamashita
2010; Yabu et al. unpubilished). The selective proteolysis of cytosolic protein substrates by CMA produces amino acids as an
energy source under the heat-shocked conditions mediated by the heat-inducible members of the HSP70 family. In addition,
macroautophagy that forms autophagic vacuoles (i.e., autophagosomes) is responsible for the survival of starved cells by
intracellular protein bulk degradation.
in the formation and activation of Activin/Nodal/TGFβ and BMP receptors.
7-2. CMA in fish
Starvation is an important stress condition that causes
a variety of physiological dysfunctions and deteriorates
the eating quality of fish. Recent studies have identified biomarkers to detect the state of stress and/or starvation in fish and cultured cells. In particular, an autophagic pathway that forms autophagic vacuoles (i.e.,
autophagosomes) is responsible for the survival of the
heat-shocked and/or starved cells by the withdrawal
of amino acids from culture medium. The autophagy
has been identified as an apoptotic pathway induced
by stress conditions, such as starvation, heat shock, and
hypoxia, followed by intracellular protein degradation
(bulk degradation), and differs from the ubiquitinproteasome system (Yabu et al. unpublished). We characterized the induction processes of autophagic pathways in cultured fish cells as a model to evaluate the
influence and the degree of biochemical changes by
heat stress and amino acid starvation (Yabu et al. unpublished). A microtubule-binding protein that is localized in autophagosome membranes can be used as
a molecular marker for induction of autophagy. We
established stable transformants of a zebrafish ZE cell
line introduced with a fusion protein of GFP and microtubule-associated protein 1-light chain 3 (MAP1LC3) to detect fluorescent autophagosomes associated
with the GFP-MAP1-LC3 fusion protein (Yabu et al.
unpublished). The induction of autophagy was examined under amino acid starvation and heat-stress conditions. The high temperature and amino acid withdrawal in the ZE cells were shown to induce autophagy
in a time-dependent manner, as evidenced by the
number of fluorescent particles localized with the GFPMAP1-LC3 fusion protein in the cell. The autophagy
induced by the amino acid withdrawal was suppressed
in the presence of the phosphatidylinositol 3-kinase
inhibitor, 3-methyladenine in the culture medium and
by overexpression of the bcl-2 gene. A
phosphatidylinositol 3-kinase, a target of rapamycin
(TOR), mediates the autophagic signaling pathway that
regulates the starvation-induced autophagy (Yabu et
al. unpublished). These results show that this is a new
biological model of the autophagic signaling pathway
in cultured fish cells under heat-stress and amino acidstarvation conditions.
The cytosolic members of the HSP70 family participate in CMA (Cuervo et al. 1995; Reggiori and
Klionsky 2002; Dice et al. 2003). The lysosomal pro-
doi:10.5047/absm.2010.00304.0111 © 2010 TERRAPUB, Tokyo. All rights reserved.
M. Yamashita et al. / Aqua-BioSci. Monogr. 4: 111–141, 2010
burnt
(heavy)
burnt
normal
D
burnt
(heavy)
burnt
normal
burnt
(heavy)
normal
burnt
(heavy)
burnt
normal
marker
C
B
A
burnt
136
Myosin heavy chain
Creatine kinase
Aldolase
HSP70/HSC70
Aldolase
Cathepsin L
Fig. 23. Induction of CMA in the muscles of bluefin tuna. The burnt tuna meat showed induction of CMA and extensive
proteolysis (Yamashita 2010). Three individuals with normal, burnt, and severely (heavy) burnt muscles were used for the
assay. Upper panel: SDS-PAGE on the water-soluble fraction. Lower panel: western blot of the lysosomal fraction obtained
by subcellular fractionation with antibodies against cathepsin L, aldolase, and HSC70. The red arrow indicates proteolytic
products found in the burnt tuna muscle. A: Internal portion in the white muscle. B: Lateral portion in the white muscle. C:
Dorsal portion of the white muscle. D: Dark muscle.
teolytic system is important in the removal of oxidized
and abnormal proteins produced under stressed conditions. In mammalian cells, three main, but different,
mechanisms contribute to the degradation of intracellular components inside lysosomes (Cuervo et al. 1995;
Reggiori and Klionsky 2002; Dice et al. 2003). HSC70
selects substrate cytosolic proteins degraded through
this pathway (Cuervo et al. 1995, 1998, 1999, 2003).
The cytosolic proteins contain a targeting motif biochemically related to the pentapeptide KFERQ (Chiang
and Dice 1988; Dice et al. 2003). This motif, present
in about 30% of the proteins in the cytosol, is recognized by HSC70. The interaction with HSC70 and with
the co-chaperones targets the substrates and leads them
to the lysosomal membrane (Agarraberes and Dice
2001). Substrates must be unfolded before translocation into the lysosomal lumen, and several cytosolic
chaperones associated with the lysosomal membrane
have been proposed to assist in the unfolding
(Agarraberes and Dice 2001). Although some basal
level of CMA is probably present in most cells, nutritional stress has been shown to maximally activate this
pathway (Cuervo et al. 1995). Activation during nutrient deprivation is associated with higher levels of
HSC70 in the lysosomal lumen (Chiang and Dice 1988;
Cuervo et al. 1995; Agarraberes et al. 1997). In addition to starvation, activation of CMA has also been
observed in rat liver and kidney following exposure to
gasoline derivatives (Cuervo et al. 1999), in fibroblasts
from patients with galactosialidosis, which lack the
protective protein/cathepsin A, and in cells
overexpressing lamp2a (Chiang and Dice 1988; Dice
et al. 2003). In contrast, CMA activity is reduced during renal tubular cell growth (Franch et al. 2001) and
in aging. The decrease in CMA activity in old cells
may be associated with their known tendency to accumulate oxidized proteins (Cuervo and Dice 2000).
The cytosolic HSP70/HSC70 might be responsible
for CMA under both normal and stressed conditions
(Yabu et al. unpublished). This effect may be depend-
doi:10.5047/absm.2010.00304.0111 © 2010 TERRAPUB, Tokyo. All rights reserved.
M. Yamashita et al. / Aqua-BioSci. Monogr. 4: 111–141, 2010
ent of the synthesis of heat-inducible HSC70 and
HSP70, which requires a minimum of 1 h under stress
conditions. The selective proteolysis of cytosolic protein substrates by CMA produces amino acids as an
energy source. The fish cells appear to utilize CMA
and to survive under the heat-shocked conditions mediated by the heat-inducible members of the HSP70
family. In mammalian cells, CMA is activated during
oxidative stress and toxic exposure resulting in the
selective degradation of chemically modified abnormal proteins altered under the stress conditions (Cuervo
et al. 1999). In goldfish GTFe-2 cells, Sato et al. (1990)
reported that a variety of stress proteins are synthesized constitutively at 37°C, with HSP70 and HSP30
being the dominantly synthesized proteins of the goldfish cells at 40°C. Therefore, enhanced CMA mediated by HSP70/HSC70 and other stress proteins may
be responsible for the tolerance to stress conditions.
HSP70/HSC70 may be used as the source of protein
degradation and thus effectively function as an energy
source under heat and other stress conditions. Therefore, in fish, HSP70 and HSC70 may play important
roles in protein degradation/catabolism by autophagy
in both stressed and non-stressed environments (Figs.
22, 23) (Yabu et al. unpublished).
Finally, the autophagic mechanism has been induced
in the muscle tissues of bluefin tuna, producing abnormal softened meat referred to as “burnt tuna meat”
(Yamashita 2010), and in chum salmon during the
spawning migration (Yamashita and Konagaya 1991).
In both cases, the muscle showed high cathepsin L activity, and the lysosomal fraction of the muscle contained HSP70/HSC70, aldolase, and cathepsin L. This
finding suggests the induced activation of CMA during and/or after the capture of fish in vivo. Therefore,
CMA may be important for understanding fish physiology as well as controlling fish meat quality under a
variety of environmental circumstances.
Acknowledgments
This work was supported in part by grants from the Fisheries Research Agency, the Japan Society of the Promotion
of Science, the Ministry of Agriculture, Forestry and Fisheries, and the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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