Regulation of heat shock genes in isolated hepatocytes from an Antarctic fish, Trematomus bernacchii
Department of Ecology, Evolution and Marine Biology, University of California, Santa Barbara, CA 93106-9610, USA
Author for correspondence (e-mail:
hofmann{at}lifesci.ucsb.edu)
Accepted 2 August 2004
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Summary |
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Key words: heat shock protein, Hsp70, hepatocyte, constitutive expression, Trematomus bernacchii, Notothenioid, Antarctica
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Introduction |
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This species may represent a rare exception to the taxonomic conservation
of the HSR. This response is an important part of the cellular defense against
proteo-toxic stress and is defined by the coordinated upregulation of several,
functionally related genes following exposure of the protein pool to
denaturing stressors such as elevated temperature or heavy metals
(Lindquist, 1986;
Parsell and Lindquist, 1993
).
The upregulated genes encode heat shock proteins (Hsps), which act as
molecular chaperones, stabilizing heat-denatured polypeptides and
disassociating unfolded proteins that have begun to interact with one another
in potentially cytotoxic aggregations
(Fink, 1999
). Those Hsps
induced by acute stress are related to isoforms of Hsps that are
constitutively expressed and perform essential chaperoning functions in the
routine synthesis of new polypeptides and the transport of proteins across
membranes (Hartl, 1996
;
Hartl and Hayer-Hartl; 2002
).
Owing to the species-independent effects of elevated temperatures on protein
structure, these genes have been maintained, at high levels of sequence
conservation, in the genomes of every eukaryotic organism examined to date
(Feder and Hofmann, 1999
). As
testament to the ecological importance of the HSR, the rapid induction of Hsps
at temperatures between 5 and 10°C above a given organism's body
temperature is nearly ubiquitous among all taxa (reviewed in
Feder and Hofmann, 1999
).
Given a habitat temperature that is constantly subzero (estimated at
1.86°C; except see Hunt et al.,
2003), an induction of Hsps in the tissues of T.
bernacchii would be expected in response to exposure to 38°C.
However, in a previous study (Hofmann et
al., 2000
), we observed no induction of Hsps in any of five
tissues that were exposed to up to 12°C, a lethal temperature for this
species (Somero and DeVries,
1967
). The constitutive isoforms of Hsps (such as Hsc71) are
present in T. bernacchii
(Carpenter and Hofmann, 2000
;
Hofmann et al., 2000
),
consistent with the important role that constitutive Hsps play in normal
cellular protein chaperoning. Interestingly, the exposure of isolated
hepatocytes to cadmium chloride, a chemical inducer of the HSR
(Lai et al., 1993
;
Ovelgonne et al., 1995
;
Liao and Freedman, 1998
), also
failed to stimulate the production of Hsps
(Hofmann et al., 2000
). This
result suggests that an anomaly exists in the expression of hsp genes
in this species, rather than insensitivity to temperature per se.
Such an anomaly could be due to numerous mechanisms, including
irregularities in the transcriptional machinery controlling Hsp expression.
All inducible size classes of Hsps are under the transcriptional control of
heat shock factor 1 (HSF1), a latent cytoplasmic transcription factor that
becomes activated through trimerization in response to stress
(Wu, 1995;
Morimoto, 1998
;
Pirkkala et al., 2001
). HSF1
binds specifically to the heat shock element (HSE), an inverted heptad repeat
(5'-nGAAn-3') in the promoters of inducible hsp genes
(Pelham, 1982
;
Xiao and Lis, 1988
). The
temperature and kinetics of HSF1 activation in poikilotherms can be dependent
upon recent thermal history (Buckley and Hofmann,
2002
,
in press
). Likewise, the Hsp
induction temperature is not fixed for a given species but varies with
acclimation or acclimatization (e.g.
Buckley et al., 2001
;
Tomanek and Somero, 2002
;
Buckley and Hofmann, 2002
).
Here, we test the following hypotheses concerning the underlying mechanism
responsible for the absence of Hsp induction in T. bernacchii. First,
the lack of protein synthesis may be due to an inability to produce stable
mRNA from hsp genes. Second, a functional HSF1 with HSE-binding
competency may be absent. Third, hsp genes may be expressed at a
constant level and are non-responsive to either heat or chemical stressors. To
test these hypotheses, we chose to study isolated hepatocytes, as this
approach allowed us to examine multiple stages of hsp gene expression
in cells from the same individual, exposed independently to a range of heat
and chemical stresses. We investigated protein synthesis patterns, Hsp70 mRNA
production and the presence, specificity and DNA-binding activity of HSF1.
Consistent with the findings of a related study from our laboratory
(Place et al., 2004), the
results support the constitutive expression of hsp genes in this
cold-adapted species.
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Materials and methods |
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Hepatocyte preparation
The preparation of hepatocytes was conducted at 1.8°C in a
temperature-controlled room (protocol after
Hofmann et al., 2000). Four
individuals were anesthetized via immersion in MS-222 (at a
concentration of 1 g l1 in seawater). The hepatic portal
vein was cannulated and the livers perfused with a buffer containing 290 mmol
l1 NaCl, 2 mmol l1 KCl, 10 mmol
l1 Hepes, 0.5 mmol l1 EGTA and 25 mmol
l1 Tricine (pH 7.8), to remove red blood cells. Livers were
then removed and incubated in a cell suspension buffer (SB) (292.5 mmol
l1 NaCl, 5 mmol l1 KCl, 2.5 mmol
l1 MgCl2, 3 mmol l1
CaCl2, 2 mmol l1 NaHCO3, 2 mmol
l1 NaH2PO4, 5 mmol
l1 glucose and 50 mmol l1 Hepes, pH 7.8)
that contained 5 units ml1 collagenase (Sigma, St Louis, MO,
USA) for 1 h, to separate cells from one another. This concentration of
collagenase was determined empirically, to dissociate cells while avoiding
cytolysis. Cells were sieved through 60 and 200 µm mesh screens and were
then pelleted via centrifugation at 100 g for 10 min.
Cells were counted on a hemocytometer and resuspended in SB at a concentration
of 3x106 cells ml1. Hepatocyte viability
was determined via bromophenol exclusion and was always >95%.
Three 1 ml aliquots of hepatocytes from each individual were exposed for 1 h to 0, 2, 4, 8 or 12°C or to 100 µmol l1 CdCl2 (Sigma) or 100 µmol l1 MG132 (Sigma). The CdCl2 and MG132 solutions were prepared in dimethylsulfoxide (DMSO). Three 1 ml aliquots were also exposed to neat DMSO as a control. Following stress exposure, for each treatment, one aliquot from each individual was assayed for (1) de novo synthesis of Hsps, (2) the presence of Hsp70 mRNA or (3) the activity of HSF1, as described below.
In vivo metabolic labeling
The de novo synthesis of protein was detected by metabolic
labeling with radiolabeled amino acids (after
Hofmann et al., 2000).
Hepatocytes were incubated with 3.7x106 Bq of
35S-labeled cysteine/methionine (specific activity
3.7x1011 mBq ml1; NEN, Torrance, CA, USA)
for 2 h at 1.8°C. Homogenates were heated at 100°C for 5 min
and centrifuged at 12 000 g for 10 min. Total counts of
incorporated radiolabel in each extract were determined on a scintillation
counter. Proteins were loaded onto 10% acrylamide gels and separated by sodium
dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE),
with an equal amount of radioactivity (75 000 c.p.m.) loaded in each lane.
Dried gels were exposed overnight to X-ray film (Kodak).
Northern blotting
Northern blots were used to assay for the presence of mRNA from both the
inducible Hsp70 and its constitutive cognate Hsc71. The probe for Hsc71 was
generated from T. bernacchii tissues using primers from trout Hsc71
(for complete description of construction of all probes, see
Place et al., 2004). Probes
for Hsp70 were constructed from heat-shocked liver tissue from two species of
notothenioid fishes, T. bernacchii and a species endemic to New
Zealand, Bovichtus variegatus. Primers were consensus sequences from
multiple alignment of several fish Hsp70 sequences. Probes were tested for
cross-reactivity with heat shock and chemically stressed hepatocytes from
T. bernacchii as well as with heat-shocked liver tissue from other
species of teleost fishes known to display typical HSRs: B.
variegatus, the New Zealand black cod (Notothenia angustata) and
Gillichthys mirabilis, a temperate, eurythermal goby. The goal was to
determine the specificity of the probes and their ability to detect the
inducible expression of Hsp70 mRNA in species possessing such a response, and
thus provide a context against which to contrast any lack of inducibility
observed in T. bernacchii. In all cases, probes detected single bands
on northern blots, with the Hsc71 band being clearly distinct from the Hsp70
band.
Following exposure to heat or chemical stress, hepatocytes were pelleted at 100 g and the supernatant discarded. Pellets were flash frozen in liquid nitrogen and stored at 80°C. Pellets were thawed in 1 ml of Trizol® reagent (Invitrogen, Carlsbad, CA, USA) and RNA extracted according to the manufacturer's protocol. For northern blotting, 10 µg total RNA was denatured with glyoxal/DMSO and blotted onto Zeta Probe nitrocellulose membrane (BioRad, Hercules, CA, USA) using a BioDot slot blotter (BioRad) under gentle vacuum. The wells were washed twice with 300 µl of 10 mmol l1 NaPO4 buffer (pH 7.0). The membranes were UV cross-linked once at 120 000 µJ cm2 using a CL-1000 UV cross-linker (UVP, Upland, CA, USA) prior to hybridization with probes.
The probes were labeled with [-32P]-dCTP
(1x106 Ci ng1 DNA; specific activity
3000 Ci mmol1) using the Ready-to-Go labeling system
(Amersham Pharmacia BioTech, Piscataway, NJ, USA). Glyoxal adducts were
removed from the membranes by incubation in 20 mmol l1
Tris-HCl (pH 8.0) at 65°C for 5 min immediately prior to prehybridization.
Membranes were prehybridized in 20 ml Church's buffer (0.5 mol
l1 NaPO4, 10 mmol l1 EDTA, 7%
SDS) at 60°C for 3 h, followed by hybridization with labeled probe at
60°C for 18 h. Following hybridization, membranes were washed twice at
room temperature for 15 min with a low-stringency wash buffer
(1xSSC/0.1% SDS) and once at 60°C for 20 min with a high-stringency
wash buffer (0.25xSSC/0.1% SDS). Following washing, membranes were
wrapped in plastic wrap and exposed to a phosphor storage screen (Molecular
Dynamics, Piscataway, NJ, USA) for 1218 h. Phosphor storage screens
were scanned using the BioRad Personal FX imager, and densitometry was
performed with Quantity One software (BioRad). Relative levels of mRNA were
background corrected and standardized across successive northern blots.
Electrophoretic mobility shift assays (EMSAs)
After the 1 h exposure, hepatocytes were pelleted by centrifugation at 100
g at 1.8°C. The supernatant was discarded and the
pellet was frozen in liquid nitrogen and stored at 80°C. Frozen
pellets were thawed in 200 µl of extract buffer containing 25% (v/v)
glycerol, 20 mmol l1 Hepes (pH 7.9), 420 mmol
l1 NaCl, 1.5 mmol l1 MgCl2, 0.2
mmol l1 EDTA, 0.5 mmol l1 PMSF and 0.5
mmol l1 dithiothreitol. Homogenized extracts were
centrifuged at 22 000 g at 4°C for 10 min. Pellets were
discarded and the supernatants frozen at 80°C, after an aliquot was
taken for total protein content determination by Bradford assay (Pierce,
Rockford, IL, USA). EMSAs were conducted according to Buckley and Hofmann
(2002). Briefly, 25 µg of
total protein from each sample was incubated, on ice, with 15 pmol of
32P-labeled oligonucleotide, containing the heat shock element
(HSE)an inverted heptad repeat sequence
(5'-GCCTCGAATGTTCGCGAAGTTT-3';
Airaksinen et al., 1998
). After
a 20 min incubation, HSF1HSE complexes were resolved on 5% acrylamide
non-denaturing gels by electrophoresis for 2 h at 250 V. Gels were dried and
exposed to a phosphoscreen. Densitometry was conducted on a scanning
phosphoimager (BioRad). All densitometry values were normalized to that of a
control run on all blots. Competitor/non-competitor assays were run to
establish the specificity of the HSE probe used in the EMSAs. In these assays,
a 200 mol l1 excess of either unlabeled HSE (competitor) or
radiolabeled AP2 oligo (non-competitor) was added to reactions containing
extract and radiolabeled HSE probe.
Statistics
The effect of heat or chemical treatment on the concentrations of Hsp70 and
Hsc71 mRNA, or on the activity of HSF1, was determined via one-way
analysis of variance (ANOVA) at a confidence level of P<0.05
(SysStat software, Point Richmond, CA, USA).
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Results |
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Hsp70 and Hsc71 mRNA analysis
Northern blotting was used to detect mRNA from Hsp70 and Hsc70 in treated
hepatocytes. Both Hsp70 and Hsc71 mRNA was detected in all individuals
(Fig. 3A). However, levels of
neither Hsp70 nor Hsc71 mRNA varied significantly among treatments
(P=0.076 and P=0.575, respectively; ANOVA;
Fig. 3B). This same Hsp70 probe
successfully detected the induction of Hsp70 in the tissues of the three other
species on which it was tested (Table
1). The same result was obtained using the second Hsp70 probe,
generated from B. variegatus
(Table 1).
|
|
HSE-binding specificity
The HSE-binding activity of HSF1 was examined in T. bernacchii, a
species for which the specificity of the HSE probe used in EMSAs had not been
previously determined. Competitor/non-competitor assays were run to determine
probe specificity, and a single HSE-specific band was observed
(Fig. 4). This band was visible
in the presence of non-competitor DNA and was absent in the presence of a 200
mol l1 excess of unlabeled competitor HSE oligonucleotide
probe.
|
HSF1 activity
EMSAs were used to determine whether the HSE-binding activity of HSF1 in
these species was responsive to temperature or chemical stressors
(Fig. 5A). While there was HSF1
present with DNA-binding activity in all samples, there was no significant
effect of treatment on HSF1 activity (Fig.
5B; P=0.23; ANOVA).
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Discussion |
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In almost all cells, an exposure to temperatures 510°C above
body temperature results in the preferential expression of multiple classes of
Hsps (Feder and Hofmann,
1999). In hepatocytes from T. bernacchii, we observed no
preferential production of any polypeptide in response to heat
(Fig. 2), although overall
protein synthesis was observed even at temperatures (812°C) that
would have been lethal to the intact organism. These results from isolated
liver cells are consistent with similar in vivo experiments on
sections of metabolically active brain, spleen, liver, heart and gill tissue
(Hofmann et al., 2000
).
Furthermore, in time-course experiments, no induction of Hsps was observed in
response to heat treatments of up to 12 h (S.P.P. and G.E.H., unpublished
data); therefore, the lack of induction here is not simply a factor of the
duration of exposure to elevated temperature. It should be noted that
two-dimensional gel electrophoresis might reveal the expression of novel
isoforms of Hsps in response to heat; however, if that is the case, the
induction of such isoforms would be minimal, as the data presented here in the
one-dimensional gels demonstrate that no significant increase in any size
class of protein was detectable.
Two chemical inducers of the HSR (CdCl2 and MG132) also failed
to increase production of Hsps (Fig.
2). Cadmium causes protein denaturation
(Abe et al., 1994) and the
creation of reactive oxygen species (Manca
et al., 1991
), while MG132 inhibits proteasome activity, resulting
in the accretion of misfolded or damaged proteins that would normally be
degraded (Bush et al., 1997
;
Lee and Goldberg, 1998
;
Kim et al., 1999
). As the
presence of abnormally unfolded proteins is a trigger of the HSR
(Hightower, 1980
;
Ananthan et al., 1986
), MG132
can be a potent chemical Hsp inducer. The inability of these chemical insults
to initiate Hsp induction supports the existence of an irregularity in the
hsp gene expression pathway rather than the loss of temperature
sensitivity in this cellular defense mechanism.
Preliminary sequencing of regions of the hsp70 gene in this
species demonstrates that this gene is present in its genome
(Maresca et al., 1988; S. Lund
and A. Whitmer, personal communication), and, in the current study, Hsp70 mRNA
was detected in isolated hepatocytes (Fig.
3A). However, we observed no treatment-related increase in the
concentration of Hsp70 mRNA (Fig.
3B), consistent with the lack of induced protein synthesis in the
in vivo metabolic labeling experiments
(Fig. 2). It should be noted
that levels of Hsp70 mRNA in treated hepatocytes were equivalent to levels in
field-caught specimens sacrificed immediately upon capture, precluding
handling stress as the cause of the observed Hsp70 mRNA expression
(Place et al., 2004
).
Furthermore, it has been demonstrated that handling stress does not affect
hepatic Hsp70 expression in other teleost species
(Vijayan et al., 1997
). The
results from the hepatocyte preparations were obtained with two Hsp70 mRNA
probes, one generated from T. bernacchii tissue and one from the
related New Zealand notothenioid B. variegatus. While both of these
probes cross-reacted with Hsp70 mRNA from T. bernacchii, in neither
case did they detect inducible expression of this transcript, although these
same probes successfully detected typical heat induction of Hsp70 mRNA in
other species of fishes (Table
1). This suggests that, despite sequence similarity in the coding
regions of this gene, transcriptional regulation of hsp70 in T.
bernacchii differs from that of these non-polar species.
To characterize the regulation of hsp genes in T.
bernacchii, we examined the DNA-binding activity of HSF1. All inducible
hsp genes are regulated by HSF1, a cytoplasmic transcription factor
that becomes activated in response to heat stress
(Wu, 1995). Inactive monomeric
HSF1 acquires the ability to bind to the HSE in hsp promoters through
trimerization and phosphorylation, a requisite step in the transactivation of
hsp genes (Morimoto,
1998
). It has been shown that cadmium stress displays a similar
mechanism of action to thermal perturbation, inducing Hsp expression through
the activation of latent monomeric HSF1
(Hung et al., 1998
;
Gordon et al., 1997
). Here, we
detected an HSE-specific HSF1 (Fig.
4).Therefore, the absence of an induction of Hsps in this species
is not due to the lack of an HSF1 with HSE-binding competency. However, the
HSE-binding activity of this molecule did not change in response to heat or
chemical treatment (Fig.
5).
The absence of inducible hsp gene expression is rare but not
unprecedented. For instance, another Antarctic organism, the ciliate
Euplotes focardii, also lacks the ability to appreciably increase the
expression of hsp70 in response to thermal stress, despite the fact
that the gene is intact in this species
(LaTerza et al., 2004). As in
T. bernacchii, hsp70 is constitutively expressed in E.
focardii (LaTerza et al.,
2001
). Taken together, these findings suggest that adaptation to
stable low temperatures may at times involve the production of molecular
chaperones at a constant level, coupled with the loss of thermal sensitivity
in the genes that encode them.
An additional species lacking a typical HSR is the freshwater hydra
Hydra oligactis, which does not induce any size class of Hsp in
response to elevated temperature and is deficient in acquired stress tolerance
(Bosch et al., 1988;
Gellner et al., 1992
). The
more thermotolerant Hydra attenuata and Hydra magnipapilata
exhibit robust stress responses and persist during periods of warming
sufficient to eliminate H. oligactis from certain environments. The
lack of an inducible HSR in H. oligactis was determined to be due not
to an inactive HSF1 or other upstream transcriptional dysfunction but rather
has been linked to the instability of Hsp mRNA in this species
(Brennecke et al., 1998
). We
cannot rule out that a similar mechanism is working synergistically with the
constitutive expression of Hsp70 mRNA in T. bernacchii, as we do not
know to what extent the message we detect is translated.
It is unknown whether other Antarctic notothenioids also lack inducible Hsp
expression. In a study using differential display, Hsp70 mRNA was induced in
the livers of another member of the suborder, the icefish Chionodraco
hamatus, in response to whole-animal injections of cadmium
(Carginale et al., 2002). The
source of the disparity between the effect of cadmium on Hsp70 induction in
T. bernacchii and that in C. hamatus is unclear but may be
due to the different methods of cadmium exposure employed. In the C.
hamatus study, cadmium was injected intramuscularly every other day for 7
days. It is possible that this longer-term exposure to the toxic heavy metal
may have a different effect than the direct exposure of liver cells conducted
here in T. bernacchii. However, it has been shown that exposure to
concentrations of cadmium as low as 5 µmol l1 are
sufficient to induce Hsp70 expression in fish cells
(Heikkila et al., 1982
). The
concentrations used here were an order of magnitude greater, yet failed to
cause Hsp induction. Another technical difference between the two studies lies
in the preparation of liver tissue. We first perfused the liver to remove red
blood cells, which are nucleated in fishes, to obtain unadulterated hepatocyte
suspensions. The C. hamatus study examined gene expression in livers
that had not been perfused, possibly resulting in the inclusion of
erythrocytes.
Our findings suggest that the hsp genes in T. bernacchii are constitutively expressed and that their transcription is regulated by an HSF1 that is active even at nearly ambient environmental temperatures. The constant expression in T. bernacchii of what are stress-inducible genes in other species may reflect an elevated need for protein chaperoning in this cold-adapted fish. Alternatively, an alteration may have occurred in the transcriptional apparatus controlling hsp genes (including but not necessarily limited to the DNA-binding activity of HSF1) that resulted in the constitutive expression of Hsps. If this is the case, the constant production of Hsps must be relatively non-deleterious. Certainly, the cold and generally uncontaminated waters of the nearshore Antarctic ecosystem may have rendered the inability to induce Hsps above a constant level selectively neutral over evolutionary time. Studies are ongoing to characterize the HSR in other species of Antarctic notothenioids, including congeners of T. bernacchii, in order to establish the extent to which the pattern of Hsp expression observed in this species is shared with other members of the suborder.
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Acknowledgments |
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Footnotes |
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