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Stress Protein HSP70 in Fish: Michiaki Yamashita, Takeshi Yabu and Nobuhiko Ojima
Stress Protein HSP70 in Fish: Michiaki Yamashita, Takeshi Yabu and Nobuhiko Ojima
111141 (2010)
www.terrapub.co.jp/onlinemonographs/absm/
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- 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
112
B: western blot
A: immunostaining
Control
HSP70
Heat shocked
Fig. 1. Expression of HSP70 in the heat-shocked zebrafish embryo. Embryos maintained at 28.5C were transferred to 37C
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.
113
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).
114
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).
115
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-
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
Heat-inducible
Heat-inducible
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.
HSE
1st exon
1st intron
117
2nd exon
ORF
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).
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)
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)
118
M. Yamashita et al. / Aqua-BioSci. Monogr. 4: 111141, 2010
119
120
DNA-BINDING DOMAIN
HEAT-ACTIVATION DOMAIN
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.
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 28.5C and heat-shocked embryos that had undergone a 1-h temperature shift to 37C, 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).
122
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 28.5C to 37C for 1 h induced hsp70 mRNA expression throughout the
entire embryo during the gastrula and segmentation periods (B), (D). Conversely, control embryos cultured at 28.5C 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 28.5C
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.
123
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.
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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 28.5C for 12 h. The control
embryos were maintained at 28.5C (control, lane 1), and the heat-shocked embryos were incubated at 37C 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.
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 37C 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
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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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
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control
As
HS
Fig. 14. Expression of the -galactosidase gene under the
128
sion under the control of the hsp70 promoter to sodium arsenite. The transgenic embryos showing -galactosidase
activity in the eyes were counted, and 50% of the fish positive for -galactosidase gene expression could be determined
at 5 M sodium arsenite (filled triangles), whereas lethality
(50% lethal dose, LD50) following a 24-h arsenite treatment
was observed at 560 M (filled circles). This finding indicates that transgene expression is a more sensitive marker
than lethality by approximately 100-fold.
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 remainsthat 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
129
Fig. 16. Transgenic zebrafish expressing green fluorescent protein (GFP) under the control of the heat-shock promoter. Heat
shock (A) and -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).
130
heat-activation
domain
1: Del1
2: Del1
3: Del2
4: Del2
5: wild-type
Del1
Del2
DNA-binding
domain
DNA-binding
domain
Del1
Del2
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 28.5C 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).
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, 1225% 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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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).
133
oventral formation.
Members of the TGF- 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- and BMP/GDF. TGF- 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; Mullera 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-
134
Ligand
Type I
receptor
Type II
receptor
active Type I
receptor
Type II
receptor
P
P
HSC/HSP70
P
P
Smad2
P-Smad2
Transcription of
target genes
Fig. 21. Model for the formation and activation of Activin/Nodal/TGF- and BMP receptors by HSP70/HSC70. HSP70/
HSC70 binds directly to the type II receptor kinases of the receptors and facilitates Nodal signaling.
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 TGF 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
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.
burnt
(heavy)
burnt
normal
D
burnt
(heavy)
burnt
normal
burnt
(heavy)
normal
burnt
(heavy)
burnt
normal
marker
burnt
136
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.
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-
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