Acute thermal stress and various heavy metals induce tissue-specific pro- or anti-apoptotic events via the p38-MAPK signal transduction pathway in Mytilus galloprovincialis (Lam.)
Department of Animal and Human Physiology, School of Biology, Faculty of Sciences, University of Athens, Panepistimioupolis, Athens 157 84, Greece
* Author for correspondence (e-mail: ibeis{at}biol.uoa.gr)
Accepted 10 October 2005
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Summary |
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Key words: mussel, copper, zinc, cadmium, signalling, apoptosis, hyperthermia, Hsp70
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Introduction |
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On the other hand, marine invertebrates such as Mytilus sp. can
face and sustain seasonal variations in environmental temperature and, as
ectotherms, they respond usually by alterations of their respiration and
metabolic rates. As an estimated proportion, 23% of the oxygen consumed
by aerobic cells is converted to oxygen radicals (O2.)
and H2O2 (Sohal and
Weindruch, 1996), and increasing tissue oxygen consumption will
entail elevated rates of ROS production in mitochondria
(Boveris and Chance, 1976
;
Boveris et al., 1976
). Higher
temperatures are therefore likely to enhance ROS release, thereby increasing
the risk of oxidative damage. ROS formation and subsequent damage are balanced
by an array of cellular antioxidant defences including various antioxidant
enzymes such as superoxide dismutase, catalase, glutathione peroxidase, as
well as low molecular binding proteins such as metallothioneins that function
as radical quenchers and as chain-breaking compounds
(Kagi and Shaffer, 1988
;
Kiningham and Kasarkis, 1998
;
Viarengo et al., 1999
).
The evaluation of oxidative stress and antioxidant balance has been
extensively studied in a plethora of cell types, tissues and animals including
marine invertebrates (for a review, see
Viarengo et al., 2000;
Molavi and Mehta, 2004
;
Vertuani et al., 2004
;
Warner et al., 2004
).
Oxidative stress induces various signal transduction pathways that lead to
either protection or apoptosis, depending on the cell type. The various signal
transduction pathways include the ones involving the mitogen-activated protein
kinases (MAPKs). The MAPK superfamily of protein Ser/Thr-kinases is a widely
distributed group of enzymes that has been highly conserved through evolution
(for reviews, see Graves and Krebs,
1999
; Widmann et al.,
1999
; Kyriakis and Avruch,
2001
; Roux and Blenis,
2004
). Three subfamilies of the MAPKs have been clearly identified
and extensively studied in mammalian experimental models: the extracellularly
responsive kinases (ERKs), the c-Jun NH2-terminal kinases (JNKs),
which are also known as the stress-activated protein kinases (SAPKs), and the
p38-MAPKs. The ERK1/2 pathway is primarily responsive to growth factors and
mitogens and appears to be involved predominantly in anabolic responses
(Widmann et al., 1999
;
Roux and Blenis, 2004
). JNKs
and p38-MAPKs are predominantly activated by various stressful stimuli and can
be involved in either anti-apoptotic or pro-apoptotic mechanisms, depending on
their isoforms and/or cell type (Widmann
et al., 1999
; Kyriakis and
Avruch, 2001
; Roux and Blenis,
2004
; Wada and Penninger,
2004
).
Among the well-established anti-apoptotic proteins, the classic,
non-ribosome-binding members of the large heat shock protein (Hsp) family are
included. These proteins exist in the cytosol of all eukaryotic cells and are
molecular chaperones required for the proper folding and trafficking of many
proteins involved in signal transduction pathways
(Buchner, 1999;
Pearl and Prodromou, 2000
). A
growing body of evidence suggests that members of the Hsp70 family are either
constitutively expressed (Hsc70) or exist as stress-inducible forms (Hsp70)
and function by binding and releasing extended polypeptide segments that are
expressed by misfolding proteins (Hartl
and Hayer-Hartl, 2002
;
Sreedhar and Csermely, 2004
).
Although the implication of Hsp70s in the anti-apoptotic molecular mechanisms
is well established, the precise signal transduction pathways leading to their
induction remain obscure.
On the other hand, the caspase-mediated apoptotic death induced by diverse
stressful conditions is well established in a plethora of mammalian cell types
(for a review, see Bredesen et al.,
2004; Jiang and Wang,
2004
; Philchenkov,
2004
). Various experimental approaches and studies have
established that oxidative stress can lead to apoptotic cell death, possibly
via cytochrome c release and the activation of various
caspases (for a review, see Jiang and
Wang, 2004
). On the contrary, very little is known on the precise
molecular mechanisms induced by environmental stress in lower vertebrates and
marine invertebrates that lead to apoptotic cell death.
In a previous paper, we had described the expression and activation of the
p38-MAPK signalling pathway in Mytilus galloprovincialis mantle
tissue in response to diverse forms of stress such as anoxia,
anoxia/re-oxygenation, oxidative stress and osmotic stress
(Gaitanaki et al., 2004). In
the present paper, we describe the effects of acute thermal stress and
indirect oxidative stress induced by various trace metals on the p38-MAPK
signal transduction pathway and its possible involvement in anti-apoptotic or
apoptotic mechanisms, depending on the cell type examined. Furthermore, we
examined the synergistic effects of thermal stress and trace metal
accumulation on the p38-MAPK signalling pathway in both mantle tissue and
gills of M. galloprovincialis.
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Materials and methods |
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Rabbit polyclonal antibodies specific for total p38-MAPK, as well as for the dually phosphorylated form of p38-MAPK, and for total Hsp70 were obtained from Cell Signalling Technology (Beverly, MA, USA). A rabbit monoclonal antibody specific for caspase-3 (#9665) that detects the endogenous levels of full-length (35 kDa) and large active fragments (17/19 kDa) of caspase-3 resulting from cleavage at Asp 175 was also purchased from Cell Signalling Technology. Prestained molecular mass markers were from New England Biolabs (Beverly, MA, USA). Biotinylated anti-rabbit antibody was from DAKO A/S (DK-2600 Glostrup, Denmark). X-OMAT AR film (13x18 cm) was purchased from Eastman Kodak Company (New York, NY, USA).
Animals
Male or female adult mussels (7580 mm length) Mytilus
galloprovincialis (Lam.) were obtained from a local dealer and had been
collected (from March up to September) in Saronikos gulf, Athens, Greece. All
animals were held in re-circulating seawater (1518°C) at the
laboratory for at least 4 days prior use.
Animal treatments
Specimens (46 for each group) were equilibrated at 15°C in large
tanks with aerated re-circulating seawater for at least 4 days. For each
treatment, animals were transferred into smaller tanks with the proper
seawater solution (300 ml per animal). Lowering the temperature down to
4°C or increasing it up to 30°C for increasing time intervals
(5120 min) induced hypothermia or hyperthermia, respectively. For the
experiments with heavy metals, either CuCl2 (1 µmol
l1), ZnCl2 (50 µmol l1) or
CdCl2 (1 µmol l1) were added to a convenient
volume of normal seawater and animals were incubated in these solutions for
increasing time intervals, varying between 15 and 120 min. In other
experiments, specimens were exposed to 1 µmol l1
CuCl2 (in normal seawater) either in the presence or absence of the
p38-MAPK selective inhibitor SB203580 (1 µmol l1).
Control experiments had shown that neither DMSO solvent nor SB203580 (1
µmol l1) alone induced any increase in p38-MAPK
phosphorylation levels.
At the end of each treatment, animals were put on ice and mantle and gill tissues were dissected, freeze-clamped between aluminium tongs cooled in liquid nitrogen, pulverised under liquid nitrogen and the powders stored at 80°C.
Tissue extractions
Mantle or gill tissue powders were homogenised with 3 ml
g1 of buffer [20 mmol l1 Hepes, pH 7.5, 20
mmol l1 ß-glycerophosphate, 20 mmol
l1 NaF, 2 mmol l1 EDTA, 0.2 mmol
l1 Na3VO4, 5 mmol l1
dithiothreitol (DTT), 10 mmol l1 benzamidine, 200 µmol
l1 leupeptin, 120 µmol l1 pepstatin A,
10 µmol l1 trans-epoxy
succinyl-L-leucylamido-(4-guanidino)butane, 300 µmol
l1 phenyl methyl sulfonyl fluoride (PMSF), 0.5% (v/v) Triton
X-100] and extracted on ice for 30 min. The samples were centrifuged (10 000
g, 5 min, 4°C) and the supernatants were boiled with 0.33
volumes of SDSPAGE sample buffer [0.33 mol l1
Tris-HCl, pH 6.8, 10% (w/v) sodium dodecyl sulphate (SDS), 13% (v/v) glycerol,
20% (v/v) 2-mercaptoethanol, 0.2% (w/v) bromophenol blue]. Protein
concentrations were determined using the BioRad Bradford assay.
SDSPAGE and immunoblot analysis
Proteins were separated by SDSPAGE on 10% (w/v) acrylamide, 0.275%
(w/v) bisacrylamide slab gels and transferred electrophoretically onto
nitrocellulose membranes (0.45 µm). Membranes were then incubated in TBS-T
[20 mmol l1 Tris-HCl, pH 7.5, 137 mmol l1
NaCl, 0.05% (v/v) Tween 20] containing 5% (w/v) non-fat milk powder for 30 min
at room temperature. Subsequently, the membranes were incubated with the
appropriate antibody according to the manufacturer's instructions. After
washing in TBS-T (3x10 min), the blots were incubated with horseradish
peroxidase-linked anti-rabbit immunoglobulin G (IgG) antibodies [1:5000
dilution in TBS-T containing 1% (w/v) non-fat milk powder; 1 h; room
temperature]. The blots were washed again in TBS-T (3x10 min) and the
bands were detected using ECL with exposure to X-OMAT AR film. Blots were
quantified by laser scanning densitometry.
DNA laddering
Extraction of high-molecular-mass DNA from the mantle and gill tissues of
M. galloprovincialis specimens was performed according to the method
described by Winnepenninckx et al.
(1993) with slight
modifications. Briefly, frozen tissue was powdered under liquid nitrogen and
homogenised with 3 ml g1 of preheated (60°C) CTAB buffer
[100 mmol l1 Tris-HCl, pH 8.0, 1.4 mol l1
NaCl, 0.2% (v/v) ß-mercaptoethanol, 2% (w/v) hexadecyltrimethylammonium
bromide-CTAB, 20 mmol l1 EDTA]. To each extract, proteinase
K (0.1 mg ml1) was added and after incubation at 60°C
for at least 30 min, the suspension was extracted with an equal volume of
phenol:chlorophorm:isoamyl alcohol (24:24:1) and centrifuged at 7700
g for 10 min at room temperature. The aqueous phase was
transferred to a new tube, extraction and centrifugation steps were repeated
and an equal volume of chlorophorm:isoamyl alcohol (24:1) was added to the
final aqueous phase. After centrifugation (7700 g, 10 min),
RNAase (30 µg ml1) was added to the aqueous phase,
samples were incubated at 37°C for 30 min and to each sample approximately
two-thirds volume of 2-propanol was added, in order to precipitate the DNA.
This was accomplished by incubating the solution overnight at room
temperature. The DNA was finally precipitated by centrifugation (7700
g, 10 min), washed in 76% (v/v) ethanol/10 mmol
l1 ammonium acetate for at least 30 min and recovered by
centrifugation (7700 g, 10 min). The samples were left to dry
in the air and the DNA was dissolved in an appropriate volume of TE buffer (10
mmol l1 Tris-HCl, pH 7.5, 0.1 mmol l1
EDTA).
The samples were diluted with water (1:200) and the degree of purity was determined by measuring the absorbance at 260 and 280 nm. Subsequently, 5 µg of DNA from each sample were loaded onto an agarose gel (1.2% w/v), along with loading buffer [0.25% (w/v) bromophenol blue, 30% (v/v) glycerol]. Electrophoresis in running buffer (445 mmol l1 Tris-HCl, pH 8.0, 445 mmol l1 boric acid, 10 mmol l1 EDTA) was performed at 70 V for approximately 4 h, the gel was treated with 4 µg ml1 ethidium bromide, observed and photographed (Fluorchem 8800; Alpha Innotech, San Leandro, CA, USA).
Activation of caspase-3
Mantle or gill tissue powders were homogenised with CHAPS buffer (1:1 w/v),
which contained 50 mmol l1 Hepes, pH 6.5, 2 mmol
l1 EDTA, 0.1% (w/v) CHAPS, 20 µg ml1
leupeptin, 10 µg ml1 pepstatin A, 10 µg
ml1 aprotinin, 5 mmol l1 DTT, 1 mmol
l1 PMSF. Following the homogenisation with a micro-pestle,
samples were frozen (80°C) and thawed, twice. The homogenates were
then centrifuged (14 000 g, 4°C, 20 min) and the
supernatants were boiled with 0.33 volumes of SDSPAGE sample buffer.
Protein concentrations were determined using the BioRad Bradford assay.
Protein samples were separated by SDSPAGE on 15% (w/v) acrylamide,
0.411% (w/v) bisacrylamide slab gels and processed for western blotting by the
use of a specific antibody raised against caspase-3, according to the
manufacturer's instructions.
Statistical evaluations
Western blots shown are representative of at least four independent
experiments. Each data point represents the mean ±
S.E.M. of samples from at least four separate
specimens treated with the respective conditions. Comparisons between control
and treatments were performed using Student's unpaired t-test. A
value of P<0.05 was considered to be statistically significant.
MAPK or caspase-3 activation and Hsp70 levels in `control' animals were set at
1, and the stimulated MAPK phosphorylation or caspase-3 activation and Hsp70
accumulation in treated animals was expressed as -fold activation over
controls.
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Results |
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From the other trace metals tested, Zn2+ at a higher concentration (50 µmol l1) induced a rapid (within 15 min) strong activation of p38-MAPK (3.19±0.02-fold relative to control values; P<0.001), reaching maximal values within 30 min (4.19±0.20-fold relative to control values; P<0.001), with a progressive decline thereafter, reaching control values within 60 min of treatment (Fig. 1). On the contrary, animal exposure to 1 µmol l1 Cd2+ induced a biphasic phosphorylation pattern of p38-MAPK in M. galloprovincialis mantle tissue. In particular, a rapid maximal phosphorylation of the kinase (6.23±0.68-fold relative to controls; P<0.001) was observed at 15 min, while a second maximum (6.89±0.20-fold relative to controls; P<0.001) was detected at 120 min of treatment (Fig. 1).
The effects of the trace metals mentioned above on the gill p38-MAPK
phosphorylation state were also examined. The results of this study showed
that, in this tissue, the phosphorylation (hence activation) of p38-MAPK
followed a qualitatively and quantitatively different profile. In particular,
1 µmol l1 CuCl2 induced a strong, rapid
phosphorylation, reaching maximum values (10.53±0.20-fold, relative to
control values; P<0.001) within 15 min, which remained elevated
for up to 60 min of treatment, with an apparent decline, although still
significantly higher than basal level, for up to 2 h oftreatment
(Fig. 3). The maximal p38-MAPK
activation obtained was comparable with that induced by 5 µmol
l1 H2O2 in M.
galloprovincialis mantle tissue
(Gaitanaki et al., 2004).
|
Cold stress as well as heat stress activated mantle tissue p38-MAPK in a time-dependent manner. Hypothermia (4°C) induced a relatively moderate phosphorylation of the kinase, with a maximum value attained within 30 min (1.76±0.13-fold, relative to control animals maintained at 15°C; P<0.05), whereas at 60 or 120 min of treatment the kinase phosphorylation levels did not differ significantly from the control values (1.63±0.34-fold or 1.44±0.23-fold, relative to control animals maintained at 15°C; P>0.05; Fig. 4A,B). Hyperthermia (30°C) also induced a rapid (within 15 min) p38-MAPK phosphorylation (2.18±0.07-fold, relative to control animals maintained at 15°C; P<0.05), reaching maximal values at 30 min (2.71±0.02-fold above basal level; P<0.01) but remaining considerably above basal values for at least 120 min (1.88±0.27-fold relative to control, P<0.05; Fig. 4A,B).
|
It is widely known that the p38-MAPK signalling pathway may be either
pro-apoptotic or anti-apoptotic depending on the kinase isoforms present in
different cell types (reviewed in Wada and
Penninger, 2004). To examine the possible involvement of this
signalling pathway in pro-apoptotic events in mantle or gill tissues under the
conditions tested, we made an effort to study the genomic DNA fragmentation
pattern in selected tissue samples. To this end, mantle and gill tissue
samples from animals treated with trace metals such as Cu2+ (1
µmol l1, for 30 min) and Zn2+ (50 µmol
l1, for 30 min) were used to isolate and examine the genomic
DNA fragmentation. The selected heavy metal concentrations as well as the
incubation time periods were identical to the ones inducing maximum activation
of p38-MAPK. The results obtained revealed that mantle and gill tissues
responded to these stressful conditions quite differently. In particular, in
the mantle tissue, both Cu2+ (1 µmol l1) and
Zn2+ (50 µmol l1) induced a rapid, extensive
increase of DNA strand breakage levels
(Fig. 5A, left panel).
Interestingly, the p38-MAPK selective inhibitor SB203580 at a concentration of
1 µmol l1 abolished the DNA fragmentation induced by 1
µmol l1 Cu2+, confirming that p38-MAPK is
involved in a pro-apoptotic signalling pathway under such conditions in the
M. galloprovincialis mantle tissue. By contrast, in gills, these two
heavy metals, as well as Cd2+ (1 µmol l1, for
15 min), induced no genomic DNA fragmentation, confirming that in this tissue
p38-MAPK is involved in an anti-apoptotic signalling pathway under such
stressful conditions (Fig. 5A,
right panel).
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In parallel, the effect of these stressful conditions on the accumulation of Hsp70 in the mantle and gill tissues was tested. The results of these experiments revealed that in gills, Cu2+ or hyperthermia (30°C) induced a significant increase of Hsp70 levels (5.14±0.45-fold and 4.21±0.50-fold relative to control values for Cu2+ and hyperthermia, respectively), whereas 1 µmol l1 SB203580 abolished the Hsp70 accumulation induced by 1 µmol l1 Cu2+, a result reconfirming the involvement of p38-MAPK in an anti-apoptotic signalling pathway in this tissue (Fig. 6). Interestingly, the combined effect of Cu2+ and hyperthermia did not result in an additive Hsp70 accumulation (Fig. 6A,B). Respective experiments on mantle tissue extracts, from animals subjected to identical stresses, showed that in this tissue there is no apparent increase in Hsp70 levels (data not shown), a result consistent with those obtained above for DNA fragmentation and caspase-3 activation (Fig. 5), indicating the induction of a possible pro-apoptotic signalling pathway.
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Discussion |
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Among the various signal transduction pathways involved in the responses to
environmental stress, MAPKs have been shown to play a significant role
(Schaeffer and Weber, 1999;
Widmann et al., 1999
;
Kyriakis and Avruch, 2001
). In
particular, p38-MAPK has been characterised as the principal stress-kinase
responsive to fluctuations in ambient osmolality and temperature
(Zhang and Cohen, 1996
;
Kultz and Burg, 1998
; Gon,
1998).
In the present study, we investigated the response of the p38-MAPK signalling pathway to acute thermal stress and various heavy metals such as Cu2+, Zn2+ and Cd2+, as well as its possible implication in either anti-apoptotic or pro-apoptotic events. Our results show, for the first time, that identical stressful conditions exemplified in vivo at the whole-animal level lead to different tissue-specific events.
As the first step in the present investigation, we examined the effect of
various heavy metals at sub-lethal concentrations on the p38-MAPK signalling
pathway in both mantle and gill tissues of M. galloprovincialis. The
results of our studies revealed that both 1 µmol l1
Cu2+ and 50 µmol l1 Zn2+ induced a
strong and transient phosphorylation (hence activation) of p38-MAPK in the
mantle tissue. Comparison of the results obtained using these two trace metals
clearly showed that mantle tissue p38-MAPK is more sensitive to
Cu2+ than to Zn2+
(Fig. 1), a result supported by
previous studies suggesting that the former induces oxidative stress
via the Fenton reaction (for a review, see
Galaris and Evangelou, 2002).
On the contrary, 1 µmol l1 Cd2+ induced a
biphasic time-dependent kinase phosphorylation profile
(Fig. 1), a result that may be
explained by the possibility that this specific kinase can be re-activated by
either its upstream or downstream substrates
(Widmann et al., 1999
;
Kyriakis and Avruch,
2001
).
Furthermore, the selective p38-MAPK inhibitor SB203580 (1 µmol
l1) was found to abolish this kinase phosphorylation induced
by 1 µmol l1 Cu2+, confirming that this
signalling pathway is specifically induced by the stressful condition examined
(Fig. 2). In addition, our
results support the suggestion that in M. galloprovincialis mantle
tissue either the or the ß1 p38-MAPK isoform exists,
since only these two are selectively inhibited by SB203580
(Goedert et al., 1997
;
Kumar et al., 1997
). Although
several investigators propose that pyridinylimidazoles inhibit the enzyme
activity rather than the phosphorylation of p38-MAPK
(Lee et al., 1994
;
Cuenda et al., 1995
), we have
clearly demonstrated that SB203580 inhibits stimulus-induced phosphorylation
of p38-MAPK. Recent studies support our finding, suggesting that this
inhibition may be due to its binding to the inactive form of the kinase,
resulting in a significant reduction of the kinase activation rate
(Frantz et al., 1998
;
Aggeli et al., 2001
;
Moro et al., 2005
).
Examination of the effects of the trace metals mentioned above on the gill p38-MAPK activation showed qualitatively and quantitatively a quite different time-dependent profile. In particular, 1 µmol l1 Cu2+ and 50 µmol l1 Zn2+ induced a strong and prolonged p38-MAPK activation for 60 min, where as 1 µmol l1 Cd2+ induced a strong, rapid activation of the kinase that remained elevated significantly above basal levels for at least 2 h (Fig. 3). The differential p38-MAPK activation by these trace metals in mantle and gill tissues may reflect their different physiological function.
Furthermore, we have found that hyperthermia (30°C) induced the
p38-MAPK activation in a rapid (maximum at 30 min), sustained (over 120 min)
and considerable way, whereas the effect of hypothermia was not so intense
(Fig. 4). The moderate response
of p38-MAPK to hypothermia in this tissue could be attributed to the fact that
these organisms are routinely subjected to hypothermic stress and have
consequently developed multiple adaptive responses in order to preserve their
function under analogous conditions
(Sheehan and Power, 1999;
Lesser and Kruse, 2004
). On
the other hand, the pleiotropic effects of heat are likely to lead to the
activation of multiple protein kinases, including p38-MAPK, which may then
regulate stress response, apoptosis or may facilitate the repair of damaged
proteins and other cellular components
(Woessmann et al., 1999
).
However, the detailed regulation of heat shock response through activation of
these signalling pathways remains to be determined.
A growing body of evidence suggests that the p38-MAPK signalling pathway is
involved in a variety of complicated cellular responses. It has been shown
that p38-MAPK cascade promotes either cell death
(Sarkar et al., 2002;
Porras et al., 2004
) or cell
survival (Liu et al., 2001
;
Park et al., 2002
), depending
on the cell type and the kinase isoforms activated by various stressful
stimuli (Kyriakis and Avruch,
2001
; Roux and Blennis, 2004;
Wada and Penninger, 2004
). In
the present study, we made an effort to examine the effect of selected
stressful conditions that induce strong p38-MAPK phosphorylation on the
caspase-3 activation, genomic DNA fragmentation and Hsp70 induction in M.
galloprovincialis mantle and gill tissues. The results of our studies
clearly demonstrate that mantle tissue responses to the heavy metals mentioned
above are quite different compared with gill tissue responses to identical
stresses. In particular, we showed for the first time that these trace metals
induce a pro-apoptotic event in the mantle tissue, possibly via the
p38-MAPK signalling pathway, since the p38-MAPK selective inhibitor abolishes
both the caspase-3 activation and the DNA fragmentation induced by such
stimuli (Fig. 5).
On the contrary, identical in vivo stimuli seem to lead to an
anti-apoptotic event in gills, via this signalling pathway, a result
that is strongly supported by the absence of DNA fragmentation and/or
caspace-3 activation (Fig. 5)
but by the induction of Hsp70 levels (Fig.
6). Our results are in accordance with the previously described
induction of metallothioneins and various antioxidant enzymes such as
superoxide dismutase and catalase by Fe in M. galloprovincialis
digestive gland (Viarengo et al.,
1999; Cavaletto et al.,
2002
).
It has also been previously reported that differences in the accumulation
of Hsp70 and other stress proteins might be useful in identifying tissues that
are particularly vulnerable to damage by a specific stressor
(Sanders et al., 1994). Our
results are in accordance with those described by Chapple et al.
(1997
), who also reported that
hyperthermia induces in gills the largest increase in Hsp70 levels, compared
with the mantle and the adductor muscle of Mytilus edulis.
Mussels have been the subject of several stress protein studies and
represent the organisms for which elevated levels of stress proteins have been
established at natural environmental temperatures
(Hofman and Somero, 1995).
Members of the Hsp70 family function as chaperones, including aiding assembly,
proper folding and the intracellular transport of proteins, thereby helping to
protect cells from thermal or other stress-induced damage
(Morimoto et al., 1990
;
Gupta and Golding, 1993
).
According to recent studies, Hsp70s antagonise the apoptosis-inducing factor,
and therefore these family members can function as potent endogenous
modulators of the apoptotic cell death
(Garrido et al., 2001
;
Zhang et al., 2002
;
Takayama et al., 2003
).
In conclusion, the results of the present study demonstrate that, in Mytilus galloprovincialis, diverse stimuli induce either pro-apoptotic or anti-apoptotic events via the p38-MAPK signalling pathway depending on the cell type examined. The differential tissue-specific responses may reflect the presence of different p38-MAPK isoforms and/or the different physiology of these tissues at the cellular and molecular level.
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Acknowledgments |
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References |
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---|
Aggeli, I.-K. S., Gaitanaki, C., Lazou, A. and Beis, I. (2001). Stimulation of multiple MAPK pathways by mechanical overload in the perfused amphibian heart. Am. J. Physiol. 281,R1689 -R1698.
Boveris, A. and Chance, B. (1976). The mitochondrial generation of hydrogen peroxide. Biochem. J. 134,707 -716.
Boveris, A., Cadenas, E. and Stoppani, A. O. M. (1976). Role of ubiquinone in the mitochondrial generation of hydrogen peroxide. Biochem. J. 156,435 -444.[Medline]
Bredesen, D. E., Mehlen, P. and Rapizadeh, S. (2004). Apoptosis and dependence receptors: a molecular basis for cellular addiction. Physiol. Res. 84,411 -430.[CrossRef]
Buchner, J. (1999). Hsp90 & Co. a holding for folding. Trends Biochem. Sci. 24,136 -141.[CrossRef][Medline]
Canesi, L., Ciacci, C., Piccoli, G., Stocchi, V., Viarengo, A. and Gallo, G. (1998). In vitro and in vivo effects of heavy metals on mussel digestive gland hexokinase activity: the role of glutathione. Comp. Biochem. Physiol. 120C,261 -268.
Cavaletto, M., Ghezzi, A., Burlando, B., Evangelisti, V., Ceratto, N. and Viarengo, A. (2002). Effect of hydrogen peroxide on antioxidant enzymes and metallothionein level in the digestive gland of Mytilus galloprovincialis. Comp. Biochem. Physiol. 131C,447 -455.
Chapple, J. P., Smerdon, G. R. and Hawkins, A. J. S. (1997). Stress-70 protein induction in Mytilus edulis: Tissue-specific responses to elevated temperature reflect relative vulnerability and physiological function. J. Exp. Mar. Biol. Ecol. 217,225 -235.[CrossRef]
Cuenda, A., Rouse, J. R., Dosa, Y. N., Meier, R., Cohen, P., Gallagher, T. F., Young, P. R. and Lee, J. C. (1995). SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1. FEBS Lett. 364,229 -231.[CrossRef][Medline]
Frantz, B., Klattm, T., Pangm, M., Parsons, J., Rolando, A., Williams, H., Tocci, M. J., O'Keefe, S. J. and O'Neill, E. A. (1998). The activation state of p38 mitogen-activated protein kinase determines the efficiency of ATP competition for pyridinylimidazole inhibitor binding. Biochemistry 37,13846 -13853.[CrossRef][Medline]
Frenzilli, G., Nigro, M., Scarcelli, V., Gorbi, S. and Regoli, F. (2001). DNA integrity and total oxyradical scavenging capacity in the Mediterranean mussel, Mytilus galloprovincialis: a field study in a highly eutrophicated coastal lagoon. Aquat. Toxicol. 53,19 -32.[CrossRef][Medline]
Gaitanaki, C., Kefaloyianni, E., Marmari, A. and Beis, I. (2004). Various stressors rapidly activate the p38-MAPK signaling pathway in Mytilus galloprovincialis (Lam.). Mol. Cell. Biochem. 260,119 -127.[CrossRef][Medline]
Galaris, D. and Evangelou, A. (2002). The role of oxidative stress in mechanisms of metal-induced carcinogenesis. Crit. Rev. Oncol. Hematol. 42, 93-103.[Medline]
Garrido, C., Gurbuxani, S., Ravagnan, L. and Kroemer, G. (2001). Heat shock proteins: endogenous modulators of apoptotic cell death. Biochem. Biophys. Res. Commun. 286,433 -442.[CrossRef][Medline]
Goedert, M., Cuenda, A., Craxton, M., Jakes, R. and Cohen,
P. (1997). Activation of the novel stress-activated protein
kinase SAPK4 by cytokines and cellular stresses is mediated by SKK3 (SKK6);
comparison of its substrate specificity with that of other SAP kinases.
EMBO J. 16,3563
-3571.
Gon, Y., Hashimoto, S., Matsumoto, K., Nakayama, T., Takeshita, I. and Takashi, H. (1998) Cooling and rewarming-induced IL-8 expression in human bronchial epithelial cells through p38 MAP kinase-depandent pathway. Biochem. Biophys. Res. Commun. 249,156 -160.[CrossRef][Medline]
Graves, J. D. and Krebs, E. G. (1999). Protein phosphorylation and signal transduction. Pharmacol. Ther. 82,111 -121.[CrossRef][Medline]
Gupta, R. S. and Golding, G. B. (1993). Evolution of HSP70 gene and its implications regulating relationships between archaebacteria, eubacteria, and eukaryotes. J. Mol. Evol. 37,573 -582.[Medline]
Hartl, F. U. and Hayer-Hartl, M. (2002).
Molecular chaperones in the cytosol: from nascent chain to folded protein.
Science 295,1852
-1858.
Hayes, J. D. and McLellan, L. I. (1999). Glutathione and glutathione-dependent enzymes represent a co-ordinately regulated defence against oxidative stress. Free Radic. Res. 31,273 -300.[Medline]
Hofman, G. E. and Somero, G. N. (1995). Evidence for protein damage at environmental temperatures: seasonal changes in levels of ubiquitin conjugates and hsp70 in the intertidal mussel Mytilus trossulus. J. Exp. Biol. 198,1509 -1518.[Medline]
Jiang, X. and Wang, X. (2004). Cytochrome c-mediated apoptosis. Annu. Rev. Biochem. 73, 87-106.[CrossRef][Medline]
Kagi, J. H. R. and Shaffer, A. (1988). Biochemistry of metallothionein. Biochemistry 27,8509 -8515.[CrossRef][Medline]
Kiningham, K. and Kasarkis, E. (1998). Antioxidant function of metallothioneins. J. Trace Elem. Exp. Med. 11,219 -226.[CrossRef]
Kultz, D. and Burg, M. (1998). Evolution of
osmotic stress signaling via MAP kinase cascades (REVIEW). J. Exp.
Biol. 201,3015
-3021.
Kumar, S., McDonnell, P. C., Gum, R. J., Hand, A. T., Lee, J. C. and Young, P. R. (1997). Novel homologues of CSBP / p38 MAP kinase: activation, substrate specificity and sensitivity to inhibition by pyridinyl imidazoles. Biochem. Biophys. Res. Commun. 235,533 -538.[CrossRef][Medline]
Kyriakis, J. M. and Avruch, J. (2001).
Mammalian mitogen-activated protein kinase signal transduction pathways
activated by stress and inflammation. Physiol. Rev.
81,807
-869.
Lee, J. C., Laydon, J. T., McDonnell, P. C., Gallagher, T. F., Kumar, S., Green, D., McNulty, D., Blumenthal, M. J., Heys, J. R., Landvatter, S. W. et al. (1994). A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature 372,739 -746.[CrossRef][Medline]
Lemaire, P. and Livingstone, D. R. (1993). Pro-oxidant/anti-oxidant processes and organic xenobiotic interactions in marine organisms, in particular the flounder Platichthys flesus and the mussel Mytilus edulis L. Trends Comp. Biochem. Physiol. 1,1119 -1149.
Lesser, M. P. and Kruse, V. A. (2004). Seasonal temperature compensation in the horse mussel, Modiolus modiolus: metabolic enzymes, oxidative stress and heat shock proteins. Comp. Biochem. Physiol. 137A,495 -504.[CrossRef]
Liu, G., Zhang, V., Bode, A. M., Ma, W. Y. and Dong, Z. (2001). Phosphorylation of 4E-BP1 is mediated by the p38/MSK1 pathway in response to UVB irradiation. J. Biol. Chem. 277,8810 -8816.[CrossRef]
Lopez-Barea, J. and Pueyo, C. (1998). Mutagen content and metabolic activation of promutagens by molluscs as biomarkers of marine pollution. Mutat. Res. 399, 3-15.[Medline]
Molavi, B. and Mehta, J. L. (2004). Oxidative stress in cardiovascular disease: molecular basis of its deleterious effects, its detection, and therapeutic consideration. Curr. Opin. Cardiol. 19,488 -493.[CrossRef][Medline]
Morimoto, R. I., Tissieres, A. and Georgopoulos, C. (1990). The stress response, function of the proteins, and perspectives. Stress Proteins Biol. Med. 1, 1-35.
Moro, T., Ogasawara, T., Chikuda, H., Ikeda, T., Ogata, N., Maruyama, Z., Komori, T., Hoshi, K., Chung, U. I., Nakamura, K. et al. (2005). Inhibition of Cdk6 expression through p38 MAP kinase is involved in differentiation of mouse prechondrocyte ATDC5. J. Cell. Physiol. 204,927 -933.[CrossRef][Medline]
Park, J. M., Greten, F. R., Li, Z. W. and Karin, M.
(2002). Macrophage apoptosis by anthrax lethal factor through p38
MAP kinase inhibition. Science
297,2048
-2051.
Pearl, L. H. and Prodromou, C. (2000). Structure and in vivo function of Hsp90. Curr. Opin. Struct. Biol. 10,46 -51.[CrossRef][Medline]
Philchenkov, A. (2004). Caspases: potential targets for regulating cell death. J. Cell. Mol. Med. 8, 432-444.[Medline]
Porras, A., Zuluaga, S., Black, E., Valladares, A., Alvarez, A.
M., Ambrosino, C., Benito, M. and Nebreda, A. R.
(2004). P38 alpha mitogen-activated protein kinase sensitises
cells to apoptosis induced by different stimuli. Mol. Biol.
Cell. 15,922
-933.
Roux, P. P. and Blenis, J. (2004). ERK and p38
MAPK-activated protein kinases: a family of protein kinases with diverse
biological functions. Microbiol. Mol. Biol. Rev.
68,320
-344.
Sarkar, D., Su, Z. Z., Lebedeva, I. V., Sanuane, M.,
Gopalkrishnan, R. V., Valerie, K., Dent, P. and Fisher, P. B.
(2002). Mda-7 (IL-24) mediates selective apoptosis in human
melanoma cells by inducing the coordinated overexpression of the GADD family
of genes by means of p38 MAPK. Proc. Natl. Acad. Sci.
USA 99,10054
-10059.
Sanders, B. M., Martin, L. S., Nelson, W. G., Hegre, E. S. and Phelps, D. K. (1994). Tissue specific stress proteins in M. edulis exposed to a range of copper concentrations. Toxicol. Appl. Pharmacol. 125,206 -213.[CrossRef][Medline]
Schaeffer, H. T. and Weber, M. J. (1999).
Mitogen-activated protein kinases: specific messages from ubiquitous
messengers. Mol. Cell. Biol.
19,2435
-2444.
Sheehan, D. and Power, A. (1999). Effects of seasonality on xenobiotic and antioxidant defence mechanisms of bivalve molluscs. Comp. Biochem. Physiol. 123C,193 -199.[CrossRef]
Sohal, R. S. and Weindruch, R. (1996). Oxidative stress, caloric restriction and aging. Science 273,59 -63.[Abstract]
Sreedhar, A. S. and Csermely, P. (2004). Heat shock proteins in the regulation of apoptosis: new strategies in tumor therapy: a comprehensive review. Pharmacol. Ther. 101,227 -257.[CrossRef][Medline]
Stohs, S. J. and Bagchi, D. (1995). Oxidative mechanisms in the toxicity of metal ions. Free Radic. Biol. Med. 18,321 -336.[CrossRef][Medline]
Takayama, S., Reed, J. and Homma, S. (2003). Heat-shock proteins as regulators of apoptosis. Oncogene 22,9041 -9047.[CrossRef][Medline]
Vertuani, S., Angusti, A. and Manfredini, S. (2004). The antioxidants and pro-antioxidants network: an overview. Curr. Pharm. Des. 10,1677 -1694.[CrossRef][Medline]
Viarengo, A. and Nott, A. (1993). Mechanisms of heavy metal cation homeostasis in marine invertebrates. Comp. Biochem. Physiol. 104C,355 -372.[CrossRef]
Viarengo, A., Arena, N., Canesi, L., Alia, F. A. and Orunesu, M. (1994). Structural and biochemical alterations in the gills of copper-exposed mussels. In Contaminants in the Environment (ed. A. Renzoni, N. Mattei, L. Lari and M.-C. Fossi), pp. 135-144. Boca Raton: Lewis Publishers.
Viarengo, A., Burlando, B., Cavaletto, M., Marchi, B., Ponzano, E. and Blasco, J. (1999). Role of metallothionein against oxidative stress in the mussel Mytilus galloprovincialis.Am. J. Physiol. 277,R1612 -R1619.[Medline]
Viarengo, A., Burlando, B., Ceratto, N. and Panfoli, I. (2000). Antioxidant role of metallothioneins: a comparative overview. Cell. Mol. Biol. 46,407 -417.[Medline]
Wada, T. and Penninger, J. M. (2004). Mitogen-activated protein kinases in apoptosis regulation. Oncogene 23,2838 -2849.[CrossRef][Medline]
Warner, D. S., Sheng, H. and Batinic-Haberle, I.
(2004). Oxidants, antioxidants and the ischemic brain.
J. Exp. Biol. 207,3221
-3231.
Widmann, C., Gibson, S., Jarpe, M. B. and Johnson, G. L.
(1999). Mitogen-activated protein kinase: conservation of a three
kinase module from yeast to human. Physiol. Rev.
79,143
-180.
Winnepenninckx, B., Backeljiau, T. and De Wachter, R. (1993). Extraction of high molecular weight DNA from molluscs. Trends Genet. 9,407 .[CrossRef][Medline]
Woessmann, W., Meng, Y. H. and Mivechi, N. F. (1999). An essential role for mitogen-activated protein kinases, ERKs, in preventing heat-induced cell death. J. Cell. Biochem. 74,648 -662.[CrossRef][Medline]
Zhang, X., Beuron, F. and Freemont, P. S. (2002). Machinery of protein folding and unfolding. Curr. Opin. Struct. Biol. 12,231 -238.[CrossRef][Medline]
Zhang, Z. and Cohen, D. M. (1996). NaCl but not urea activates p38 and jun kinase in mIMCD3 murine inner medullary cells. Am. J. Physiol. 271,F1234 -F1238.[Medline]