Download Noradrenaline and hsp70 expression in mollusc immune cells

RESEARCH ARTICLE
3557
Noradrenaline and α-adrenergic signaling induce the
hsp70 gene promoter in mollusc immune cells
Arnaud Lacoste*, Marie-Cécile De Cian, Anne Cueff and Serge A. Poulet
Station Biologique de Roscoff, CNRS – Université Paris VI – INSU, Place Georges Teissier, BP 74, F-29682 Roscoff Cedex, France
*Author for correspondence (e-mail: [email protected])
Accepted 18 June 2001
Journal of Cell Science 114, 3557-3564 © The Company of Biologists Ltd
SUMMARY
Expression of heat shock proteins (hsp) is a homeostatic
mechanism induced in both prokaryotic and eukaryotic
cells in response to metabolic and environmental insults. A
growing body of evidence suggests that in mammals, the
hsp response is integrated with physiological responses
through neuroendocrine signaling. In the present study, we
have examined the effect of noradrenaline (NA) on the
hsp70 response in mollusc immune cells. Oyster and
abalone hemocytes transfected with a gene construct
containing a gastropod hsp70 gene promoter linked to the
luciferase reporter-gene were exposed to physiological
concentrations of NA, or to various α- and β-adrenoceptor
agonists and antagonists. Results show that NA and
α-adrenergic stimulations induced the expression of
luciferase
in
transfected
mollusc
immunocytes.
Furthermore, exposure of hemocytes to NA or to the αINTRODUCTION
The induction of ‘heat shock’ or ‘stress’ proteins represents a
homeostatic defense mechanism of cells in response to
metabolic and environmental insults. Heat shock proteins (hsp)
are encoded by a family of highly conserved genes present in
both eukaryotic and prokaryotic cells and range in size from
10 to 110 kDa, with the 70 kDa hsp (hsp70) being the most
abundant and best-characterized members of this protein
family. Studies on the expression of hsp have provided
evidence of a complex pattern of regulation. Indeed, induction
of hsp results from a variety of stressors including elevated
temperatures, exposure to heavy metals or amino acid
analogues, presence of eukaryotic parasites and viral infection.
Some hsp genes are regulated in a cell-cycle-dependent
manner; most hsp are also constitutively expressed in normal
unstressed cells and function as molecular chaperones in
protein biosynthesis to facilitate protein folding, assembly,
secretion, regulation, degradation and translocation (Lindquist
and Craig, 1988; Feder and Hofmann, 1999).
Most attention has been focused on the functions of hsp at
the molecular and cellular levels. Questions are now emerging
on how the hsp response is integrated with fundamental
physiological stress responses at the animal level (Feder
and Hofmann, 1999). Previous studies have shown that in
vertebrates, restraint-stress induces an hsp response in the
adrenal gland and the aorta that is dependent on the activation
of the hypothalamic-pituitary-adrenal axis and sympathetic
adrenoceptor agonist phenylephrine (PE) resulted in the
expression of the inducible isoform of the hsp70 protein.
Pertussis toxin (PTX), the phospholipase C (PLC) inhibitor
U73122, the protein kinase C (PKC) inhibitor calphostin C,
the Ca2+-dependent PKC inhibitor Gö 6976 and the
phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor
LY294002 blocked the PE-mediated induction of the hsp70
gene promoter. These results suggest that α-adrenergic
signaling induces the transcriptionnal upregulation of
hsp70 in mollusc hemocytes through a PTX-sensitive Gprotein, PLC, Ca2+-dependent PKC and PI 3-kinase. Thus,
a functional link exists between neuroendocrine signaling
and the hsp70 response in mollusc immune cells.
Key words: Mollusc, Immune cell, Noradrenaline, Heat shock
protein, α-adrenergic signaling, Thermotolerance
nervous system (Blake et al., 1991; Udelsman et al., 1993).
Moreover, perturbation of the hypothalamic-pituitary-adrenal
axis results in induction of hsp70 in several rat tissues
(Udelsman et al., 1994).
In molluscs, stress also results both in physiological
responses – such as the secretion of catecholamines (Lacoste
et al., 2001a) – and hsp responses (Hofmann and Somero,
1995; Hofmann and Somero, 1996; Clegg et al., 1998; Werner
and Hinton, 1999; Feder and Hofmann, 1999; Minier et al.,
2000). Although catecholamines are known to play essential
roles in several physiological processes in molluscs including
feeding (Teyke et al., 1993), locomotion (Sakharov and
Salànski, 1982), respiration (Syed and Winlow, 1991),
reproduction (Martínez and Rivera, 1994) and development
(Pires et al., 1997), data are lacking concerning the effects of
these hormones on the expression of hsp in mollusc cells.
Oyster hemocytes, a category of cells that constitute a primary
line of defense against invasive pathogens and parasites, have
the ability to elicit an hsp response which, supposedly, enables
them to maintain immune surveillance during or after stressful
events that threaten the animal’s survival (Tirard et al., 1995).
Secretion of noradrenaline (NA) also occurs in response to
stress in molluscs (Lacoste et al., 2001a) and this
catecholamine has recently been shown to modulate oyster
hemocyte functions (Lacoste et al., 2001b). In the present
study, we have used transfection techniques and a gene
construct containing a gastropod hsp70 gene promoter linked
to the luciferase reporter-gene to determine the effect of NA
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JOURNAL OF CELL SCIENCE 114 (19)
on the expression of the hsp70 gene in mollusc immune cells.
This approach, which has proved efficient for the study of hsp
gene expression in both vertebrates and invertebrates (Roigas
et al., 1997; Akagawa et al., 1999; Link et al., 1999; Adam et
al., 2000), has allowed us to elucidate signal transduction
pathways involved in the NA-mediated induction of the hsp70
gene promoter in mollusc hemocytes.
MATERIALS AND METHODS
Drugs
NA, the α-adrenoceptor agonist phenylephrine (PE), the βadrenoceptor agonist isoproterenol, the α-adrenoceptor antagonist
prazosin, the β-adrenoceptor antagonist propanolol, pertussis toxin
(PTX), the phospholipase C (PLC) inhibitor U73122, the protein
kinase C (PKC)-specific inhibitor calphostin C, the protein kinase A
(PKA) specific inhibitor H-89, the phosphatidylinositol-3 kinase (PI
3-kinase) inhibitor LY294002 and the mitogen-activated protein
(MAP) kinase kinase inhibitor PD059098 were all obtained from
Sigma. The PKC inhibitor Gö 6976 was purchased from Calbiochem.
Oyster and abalone hemocytes
Oysters Crassostrea gigas and abalones Haliotis tuberculata were
maintained in polyethylene tanks containing 110 l of aerated and
continuously flowing (50 l/hour) natural seawater at 14-15°C.
Animals were left undisturbed for a 10 day acclimation period before
experiments. Hemolymph (0.5-1 ml/oyster or 2-3 ml/abalone) was
collected from the pericardial cavity using 2 ml syringes and 21 G×1.5
inch needles. Hemolymph was pooled to obtain 10-15 ml samples.
Hemocyte concentration was determined using a hemacytometer and
adjusted to 106 cell/ml by the addition of modified Hank’s balanced
salt solution (MHBSS) consisting of HBSS adjusted to ambient sea
water salinity (31 ppm) and pH 7.4 and containing 2 g/l of D-glucose,
110 mg/l of sodium pyruvate (Gibco) and 55 mg/l of ascorbic acid
(Sigma).
Transfection
Protocols used for hemocyte transfection were inspired from previous
studies showing that cationic lipids allow foreign gene transfer into
mollusc cells (Yoshino et al., 1998; Cadoret et al., 1999). The reportergene construct used in the present study has been described previously
(Yoshino et al., 1998). It consists of the gastropod Biomphalaria
glabrata hsp70 gene promoter cloned just upstream of the firefly
Photinus pyralis luciferase reporter-gene in the pSP-Luc+ vector
(Promega). Control constructs contained the hsp70 gene promoter
alone or the luciferase reporter-gene alone. For transfection, hemocyte
suspensions were divided into 2 ml aliquots and left to attach in 35
mm Petri dishes (2.106 cells/dish) for 20 minutes, rinsed with MHBSS
and incubated for 1 hour at 17°C in MHBSS containing 20% DMEM
(Gibco) adjusted to ambient salinity (31 ppm). Cells were then rinsed
twice in MHBSS containing 20% DMEM and exposed for 2 hours at
17°C to a 1:5 mixture of 10 µg DNA precomplexed to Plus reagent
(Gibco) and lipofectamine (Gibco) in 1ml MHBSS containing 20%
DMEM. To increase transfection efficiency, a multiple transfection
protocol (Yamamoto et al., 1999) was used (transfection was repeated
for a total of four times over an 8 hour period). The volume of medium
was then increased to 3 ml by the addition of modified IMDM (Gibco)
adjusted to ambient salinity (31 ppm) and containing 5% horse serum,
5% fetal bovine serum, penicillin G (50 units/ ml), streptomycin (50
µg/ml) and NA, PE or isoproterenol at concentrations indicated in
Results. In some experiments, antagonists or inhibitors were added 30
minutes (or 6 hours in the case of PTX) prior to the addition of NA
or PE. Cells were then incubated in the presence of the various drugs
for 24 hours before luciferase activity was measured. In experiments
where heat shock treatment was given, the cells were incubated at
41°C for 60 minutes followed by incubation for 24 hours at 17°C for
the expression of luciferase.
Measurement of luciferase activity
At the end of the incubation period, the medium was carefully
removed and cells were lysed in 100 µl of cell lysis buffer provided
with the Promega luciferase assay system. Cell lysates were
transferred to microcentrifuge tubes and immediately frozen at −80°C.
For luciferase activity measurements, samples were thawed on ice and
centrifuged at 12,000 g for 2 minutes at 4°C. Fifty microliters of
sample were then transferred to luminometer tubes containing 100 µl
of luciferase assay reagent (Promega). Light emission was measured
using a Lumat LB 9507 luminometer (E. G. & G. Berthold) and data
were expressed as relative light unit (RLU)/mg protein/minute.
Sample protein concentrations were determined using the Bradford
method (Bradford, 1976) with bovine serum albumin as the protein
standard.
Immunoblot assays
Western blots were performed on protein extracts originating from
oyster hemocytes incubated in the presence of either NA, PE or
isoproterenol for 24 hours or exposed to 41°C for 60 minutes followed
by incubation for 24 hours at 17°C. Cells were washed in MHBSS
and lysed by sonication for 1 minute at 20-25 mA (VC 75455
sonicator, Bioblock Scientific) in 50 mM Tris-HCl, pH 6.8 containing
2 mM EDTA, 200 mM sucrose, 150 mM KCl, 5 mg/ml chymostatin,
10 mg/ml aprotinin, 10 mg/ml leupeptin and 25 mg/ml 4-(2aminoethyl)benzenesulfonyl fluoride (AEBSF) (all from Sigma).
Samples were then centrifuged at 10,000 g for 30 minutes and aliquots
of 50 µg protein extracts were boiled at 100°C for 5 minutes,
separated onto 12% LiDS-polyacrylamide gels and transferred to
nitrocellulose membranes (Protran BA 83, Schleicher & Schuell) as
described by Towbin et al. (Towbin et al., 1979). Blots were then
probed with a 1:3000 dilution of a mouse anti-human hsp70 antibody
(Affinity Bioreagents), which is known to recognize both constitutive
and inducible hsp70 isoforms in a wide range of vertebrate and
invertebrate species including oysters (Tirard et al., 1995). The
secondary antibody was a horseradish peroxidase-conjugated goat
anti-mouse IgG (Biorad) at a 1:3000 dilution. Labelled proteins were
detected with an enhanced chemiluminescence reagent (100 mM TrisHCl, pH 8.5 containing 0.01% hydrogen peroxyde, 1.25 µM luminol
and 0.23 µM coumaric acid) and X-Omat AR Kodak Scientific
Imaging films.
Thermotolerance assay
Cells were incubated for 24 hours at 17°C in 300 µl modified IMDM
alone or IMDM containing NA, PE or isoproterenol at concentrations
indicated in the text. Samples were then incubated for 60 minutes at
45°C. This temperature approaches the thermal threshold (47-48°C)
after which oyster hemocyte viability and cellular metabolism are
not detected (Tirard et al., 1995), thus it was more suitable for
thermotolerance assays than the 41°C heat stress used in other
experiments as an optimal temperature for the induction of luciferase
expression. After the heat treatment, hemocytes were returned to
17°C for 6 hours. The number of viable metabolically active cells
was then determined using a 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)
tetrazolium bioreduction assay (Promega) according to the
manufacturer’s instructions. Briefly, 60 µl of MTS One-Step Solution
(Promega) were added to the medium and samples were incubated
for 2 hours at 17°C. The quantity of formazan product, which is
directly proportional to the number of viable metabolically active
cells, was then determined by recording absorbance at 490 nm.
Results were expressed as percentage of viable cells.
Statistical analyses
Data are presented as means and standard errors of at least three
Noradrenaline and hsp70 expression in mollusc immune cells
25
15
hsp-luc
hsp only
luc only
10
A
**
**
Luciferase activity
(RLU / mg protein)
(RLU / mg protein)
Luciferase activity
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*
20
10
5
5
0
0
0.1
1.0
10.0
0
Phenylephrine (µM)
0.1
1. 0
10
10.0
**
B
**
Luciferase activity
(RLU / mg protein)
0
20
C
15
10
*
*
5
0
NA
1
10
100
Prazosin (µM) + NA
0
0.1
1. 0
10.0
Noradrenaline (µM)
Fig. 1. Noradrenaline induces the hsp70 gene promoter in oyster
Crassostrea gigas and abalone Haliotis tuberculata immune cells.
Hemocytes transfected with a gene construct containing the luciferase
reporter-gene under the transcriptional control of the mollusc hsp70
gene promoter (hsp-luc) were exposed to NA for 24 hours. Exposure
to NA resulted in increased luciferase activity in both oyster (A) and
abalone (B) hemocytes. Luciferase activity was however almost twice
as high in oyster than in abalone hemocytes, which is probably due to
lower transfection efficiency and cell viability in abalone hemocytes.
Constructs containing the hsp70 gene promoter alone (hsp only) or
the luciferase gene alone (luc only) were used as controls. Data are
means and standard errors of three replicate experiments. Asterisks
denote significant (* for P<0.05, ** for P<0.01) differences from
samples incubated in the absence of NA.
experiments. For comparison of two means, paired or unpaired t-tests
were used where appropriate. For multiple comparisons, the data were
analyzed by one-way analysis of variance. Unless otherwise indicated,
P<0.01 was considered as the limit of significance.
RESULTS
A reporter construct expressing the luciferase luc gene under
the control of the mollusc hsp70 gene promoter was
transfected into hemocytes of the bivalve Crassostrea gigas
and the gastropod Haliotis tuberculata. Results in Fig. 1 show
that a 24 hour exposure to NA at concentrations ranging
between 0.1 and 10 µM resulted in a significant expression of
luciferase in both oyster (Fig. 1A) and abalone (Fig. 1B)
hemocytes, suggesting that NA induces the hsp70 gene
D
10
5
Bas
5
0.1
1.0
10.0
Isoproterenol (µM)
15
0
0
B
15
*
10
5
25
20
15
20
(RLU / mg protein)
*
*
0
0
Luciferase activity
A
3559
Bas
NA
1
10
100
Propanolol (µM) + NA
Fig. 2. α-Adrenergic stimulation but not β-adrenergic stimulation
induces the hsp70 gene promoter in oyster immune cells. Luciferase
activity increased in transfected cells exposed to the α-adrenoceptor
agonist PE (A), but not in cells exposed to the β-adrenoceptor
agonist isoproterenol (B). Pretreatment with the α-adrenoceptor
antagonist prazosin blocked the NA-induced increase in luciferase
activity (C), whereas pretreatment with the β-adrenoceptor
antagonist propanolol had no significant effect (D). Data are means
and standard errors of three replicate experiments. Asterisks denote
significant (P<0.01) differences from samples incubated in the
absence of agonist (A,B) or in the presence of 1 µM NA alone (C,D).
Bas indicates luciferase activity in samples incubated in the absence
of drugs.
promoter in these cellular systems. Luciferase activity was,
however, almost twice as high in oyster hemocytes than
abalone hemocytes, which is probably due to lower
transfection efficiency and cell viability in abalone hemocytes.
For this reason, subsequent experiments were performed on
oysters cells only. Control constructs containing the hsp70
gene promoter alone, or the luciferase reporter-gene alone, did
not result in any significant increase in luminescence or
luciferase activity (Fig. 1A,B).
To determine the type of adrenoceptor responsible for the
induction of the hsp70 promoter, the effects of PE (an αadrenoceptor agonist) and isoproterenol (a β-adrenoceptor
agonist) were tested. A 24 hour exposure to PE resulted in
significantly (P<0.01) higher luciferase activity in oyster
hemocytes compared with controls (Fig. 2A). Isoproterenol
had no significant (P>0.01) effect on the expression of
luciferase (Fig. 2B). Moreover, the α-adrenoceptor antagonist
prazosin blocked the inducing effect of NA on the hsp70 gene
promoter (Fig. 2C), whereas the β-adrenoceptor antagonist
propanolol had no significant (P>0.01) effect (Fig. 2D).
Measurement of hsp70 protein synthesis by means of
immunoblot assays (Fig. 3) showed that a 24 hour exposure to
1-10 µM NA and 1-10 µM PE induced the expression of the
inducible isoform of hsp70 proteins, whereas in the presence
of 0.1-10 µM isoproterenol, only the constitutive isoform was
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JOURNAL OF CELL SCIENCE 114 (19)
Fig. 3. Noradrenaline and the α-adrenoceptor agonist PE induce
expression of the inducible hsp70 isoform (lower band) in oyster
immune cells, whereas in cells exposed to the β-adrenoceptor agonist
isoproterenol (ISO), the constitutive hsp70 protein (upper band) is the
only isoform expressed. Untreated control samples (CT) permit
visualisation of the constitutive oyster hsp70 isoform band, whereas samples originating from heat-shocked cells (HS) show bands
corresponding to both constitutive and inducible oyster hsp70 proteins. Reference proteins were rabbit muscle phosphorylase b (97.4 kDa) and
bovine serum albumin (66.2 kDa).
20
A
15
20
B
15
*
10
*
5
5
0
0
Bas
PE
1
10
*
10
100
Bas
PE
PTX (ng/ml) + PE
Luciferase activity
(RLU / mg protein)
20
15
1
10
*
5
D
PE
H-89
CalC
*
5
PE
0.1
1
10
LY294002 (µM) + PE
*
10
*
*
0
Bas
10
Bas
5
0
*
0
15
10
15
U73122 (µM) + PE
20
C
0.1
*
Luciferase activity
(RLU / mg protein)
Luciferase activity
(RLU / mg protein)
20
Bas
PE
0.1
1
10
..
Go 6976 (µM) + PE
Fig. 4. α-Adrenergic induction of the hsp70 gene involves a PTXsensitive G-protein, PLC and a Ca2+-dependent PKC. Pretreatment of
transfected oyster hemocytes with (A) PTX, (B) the PLC inhibitor
U73122, (C) the PKC inhibitor calphostin C (CalC) and (D) the
Ca2+-dependent PKC isoform inhibitor Gö 6976 blocked the PEinduced increase in luciferase activity. Pretreatment with the PKA
inhibitor H-89 (C) had no significant effect. Data are means and
standard errors of three replicate experiments. Asterisks denote
significant (P<0.01) differences from samples incubated in the
presence of 1 µM PE alone. Bas indicates luciferase activity in
samples incubated in the absence of drugs.
expressed. These results suggest that NA acts through αadrenoceptors to induce the hsp70 gene promoter in oyster
hemocytes.
Further experiments were conducted to elucidate some of the
signal transduction pathways involved in the α-adrenergic
induction of the hsp70 gene promoter in oyster immune cells.
Results in Fig. 4A show that 10 and 100 ng/ml of PTX
significantly (P<0.01) blocked the PE-mediated induction of
luciferase expression. In the presence of ≥1 µM of the PLC
inhibitor U73122, the PE-activated expression of luciferase
was also blocked (Fig. 4B). Moreover, the PKA inhibitor H-89
(25 µM) had no significant (P>0.01) effect on the induction of
luciferase activity by PE, whereas the PKC inhibitor calphostin
C (80 nM) significantly (P<0.01) attenuated the inducing effect
of the α-adrenoceptor agonist (Fig. 4C). Gö 6976, which
selectively inhibits Ca2+-dependent PKCα- and PKC β1isozymes in mammals, also blocked the activation of luciferase
Fig. 5. α-Adrenergic induction of the hsp70 gene involves a PI 3kinase. Pretreatment of transfected oyster hemocytes with the PI 3kinase inhibitor LY294002 blocked the PE-induced increase in
luciferase activity. Data are means and standard errors of three
replicate experiments. Asterisks denote significant (P<0.01)
differences from samples incubated in the presence of 1 µM PE
alone. Bas indicates luciferase activity in samples incubated in the
absence of drugs.
activity by PE (Fig. 4D). From these results we conclude that
α-adrenoceptor-mediated induction of the molluscan hsp70
gene promoter involves a PTX-sensitive G-protein, PLC and
Ca2+-dependent PKC isoforms.
Recent studies have suggested that PI 3-kinases act as
second messengers and regulate PLC-mediated calcium
signaling in vertebrate cells (Rameh et al., 1998). To determine
whether PI 3-kinase is required for the α-adrenoceptormediated induction of the hsp70 gene promoter in mollusc
hemocytes, the specific PI 3-kinase inhibitor LY294002 was
used. Results in Fig. 5 show that at concentrations ≥1 µM,
LY294002 significantly blocked the induction of luciferase
activity by PE.
By contrast, at concentrations ≤50 µM, the MAP kinase
kinase inhibitor PD098059 had no significant (P>0.01) effect
on the PE-induced stimulation of luciferase activity (Fig. 6),
indicating that activation of the MAP kinase cascade is not
required for the α-adrenergic induction of the hsp70 gene
promoter in oyster hemocytes. At a concentration of 100 µM,
PD098059 resulted in higher luciferase activity in both PEtreated and PE-untreated hemocytes, suggesting that the MAP
kinase cascade may repress the induction of the hsp70 gene
promoter in oyster immune cells.
Finally, experiments were conducted to determine the effects
of NA and α- and β-adrenoceptor agonists on the hsp70
response to heat shock in oyster immune cells. Results in Fig.
7A show that a 24 hour exposure to 1 µM NA or PE prior to
Noradrenaline and hsp70 expression in mollusc immune cells
*
20
40
15
10
**
5
Luciferase activity
(RLU / mg protein)
Luciferase activity
(RLU / mg protein)
25
0
No Heat Stress
Heat Stress bb
bb
3561
A
30
aa
20
aa
10
0
Bas
PE
10
50
100
Bas
PD098059 (µM)
PE + PD
PD alone
heat shock resulted in a significantly (P<0.01) higher luciferase
activity compared to oyster immune cells incubated in the
absence of drugs (Bas) before exposure to heat shock.
Isoproterenol tended to cause lower luciferase activity in
hemocytes submitted to high temperature; however, this
difference was not significant (P>0.01). In addition,
thermotolerance assays (Fig. 7B) showed that exposure to NA
or PE resulted in higher viability in hemocytes submitted to
severe heat stress (45°C for 60 minutes), suggesting that αadrenergic stimulation leads to higher thermotolerance in these
cells.
DISCUSSION
Transcriptional upregulation of hsp is a common component of
the cellular response to stress. An effective technique for the
examination of regulatory mechanisms controlling gene
transcription is the introduction of reporter-gene constructs into
cells of interest. This approach has notably proven effectual in
the study of hsp gene regulation in a number of vertebrates and
invertebrates including mammals (Chu et al., 1996; Roigas et
al., 1997; Akagawa et al., 1999), Xenopus (Krone and Heikkila,
1989), zebrafish (Adam et al., 2000) and the nematode
Caenorhabditis elegans (Link et al., 1999). In the present
study, we used a gene construct containing the luciferase
reporter-gene under the transcriptionnal control of a gastropod
hsp70 gene promoter to determine the effect of NA and
adrenergic stimulation on hsp70 gene expression in mollusc
immune cells. In higher eukaryotes, expression of heat shock
80
b
% viable cells
Fig. 6. α-Adrenergic induction of the hsp70 gene does not involve
the MAP kinase cascade. Pretreatment of transfected oyster
hemocytes with the MAP kinase inhibitor PD098059 (PD) did not
block the PE-induced increase in luciferase activity. Exposure of
cells to 100 µM PD098059 resulted in increased luciferase activity in
both PE-treated and -untreated samples, suggesting that MAP
kinases repress the induction of the hsp70 gene promoter in oyster
immune cells. Data are means and standard errors of three replicate
experiments. Asterisks denote significant (* for P<0.05, ** for
P<0.01) differences from samples incubated in the absence of drugs
(white bars) or in the presence 1 µM PE alone (hatched bars). Bas
indicates luciferase activity in samples incubated in the absence of
drugs.
NA
PE
ISO
bb
B
60
40
20
0
Bas
NA
PE
ISO
Fig. 7. Noradrenaline and PE but not isoproterenol (ISO) potentiate
the induction of the hsp70 gene promoter and increase
thermotolerance in oyster immune cells subjected to heat-shock.
(A) The heat (41°C)-induced increase in luciferase activity is
significantly higher in oyster hemocytes pretreated with NA or PE
but not in cells pretreated with isoproterenol. (B) Cell survival after
severe heat-treatment (45°C, 60 minutes) is significantly higher in
oyster hemocytes pretreated with NA or PE than in control samples
incubated in the absence of drugs (Bas). Cell survival is not
significantly different in cells pretreated with isoproterenol. Data are
means and standard errors of three replicate experiments. (a) and (b)
above the error bars denote significant (one letter for P<0.05, two
letters for P<0.01) differences from non-heat-shocked samples (a) or
heat-shocked samples (b) incubated in the absence of drugs (Bas).
genes is regulated at the transcriptional level by the specific
heat shock transcription factors (HSF) which, upon activation,
trimerize and bind to heat shock elements (HSE) present in the
promoter region of heat shock genes (Wu, 1995). Although
HSF have not yet been described in molluscs, the gastropod
hsp70 gene promoter used in this study comprises several HSE
and other features common to vertebrate hsp70 gene promoters
including TATA boxes and CAAT sequences which, in
conjunction to HSE, are thought to function as enhancers of
hsp gene induction and confer its heat-inducibility to the
promoter (Yoshino et al., 1998).
Our results demonstrate that exposure to NA leads to the
induction of the hsp70 gene promoter in both gastropod and
bivalve immune cells (Fig. 1). The NA concentrations used in
the present study fall within ranges reported in mollusc tissues
and hemolymph (Osada and Nomura, 1989; Pani and Croll,
1995; Lacoste et al., 2001a). Furthermore, they exert
physiological effects in molluscs (Muneoka and Kamura,
1982; Coon and Bonar, 1987; Lacoste et al., 2001b), suggesting
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JOURNAL OF CELL SCIENCE 114 (19)
that they are of relevance in vivo. As a consequence, NAmediated transcriptional upregulation of hsp70 may occur, for
example, when molluscs face stressful environmental
situations that trigger the release of NA in the hemolymph.
The use of α- and β-adrenoceptor agonists and antagonists
showed that the NA-mediated induction of the hsp70 gene
promoter and of hsp70 protein synthesis involves αadrenoceptors (Fig. 2; Fig. 3). Previous studies have shown that
α-adrenergic regulation mediates metamorphosis in molluscs
(Coon and Bonar, 1987) and we have recently provided
evidence for the presence of β-adrenoceptors in oyster
hemocytes (Lacoste et al., 2001b). However, the present results
show for the first time that α-adrenoceptors are present at the
surface of mollusc immune cells.
In oyster hemocytes, α-adrenoceptors couple to a PTXsensitive G-protein to mediate the induction of the hsp70 gene
promoter (Fig. 4). This result is not consistent with the
generally accepted idea that vertebrate α-adrenoceptorstimulated responses are predominantly mediated by PTXinsensitive G-proteins, likely the Gq family. However, it is
coherent with other studies showing that PTX-sensitive Gproteins can be utilized to transduce α-adrenergic stimulation
(Perez et al., 1993). Interestingly, another study has
demonstrated the existence of a β-adrenoceptor/PTX-sensitive
G-protein in mollusc sarcolemma rather than the βadrenoceptor/PTX-insensitive G-protein functional coupling
characteristic of vertebrates (Pertseva et al., 1992). Information
on associations between adrenoceptors and G-proteins in
molluscs are scarce, hence it is not possible to determine
whether mollusc adrenoceptors usually couple to PTXsensitive G-proteins rather than to PTX-insensitive G-proteins;
however, this topic deserves further attention.
Induction of the hsp70 gene promoter through α-adrenergic
signaling was also found to involve PLC and PKC, which is
consistent with previous studies showing that the activation of
α-adrenoceptors stimulate PLC in mammals (Cohen and
Almazan, 1993) and that PKC-responsive signaling pathways
are involved in the regulation of the heat shock response in
human cells (Erdos and Lee, 1994; Holmberg et al., 1997;
Holmberg et al., 1998). Our results are also consistent with
recent studies suggesting that serotonin, another biogenic
amine present in molluscs, may function through receptors
linked to PKC in Aplysia (Fox and Lloyd, 2000). Interestingly,
the indocarbazole Gö 6976, which selectively inhibits
Ca2+-dependent PKCα and PKCβ1 isozymes in vertebrates
(Martiny-Baron et al., 1993), blocked the α-adrenergic
induction of the hsp70 promoter gene in oyster immune cells.
Although the existence of Ca2+-dependent PKCs have been
demonstrated previously in Aplysia (Nakhost et al., 1998), our
results provide the first evidence for Gö 6976-sensitive PKC
isoforms in an invertebrate.
Activated PKCs in turn phosphorylate a wide range of
effector proteins. We focused on two kinase families that are
activated by PKC and α-adrenergic stimulation: PI 3-kinases
and MAP kinases. PI 3-kinases are lipid kinases that
phosphorylate phosphatidylinositol 4,5-biphosphate to
phosphatidylinositol 3,4,5-triphosphate. Growing evidence
suggests that PI 3-kinases play important roles in signal
transduction (Downward, 1998). For example, αadrenoceptors activate PI 3-kinases in human vascular smooth
muscle cells (Hu et al., 1996) and PI 3-kinases regulate PLC-
mediated calcium signaling (Rameh, 1998). LY294002, a
highly specific inhibitor of PI3-kinases (Vlahos et al., 1994),
blocked the α-adrenergic induction of luciferase activity in our
transient expression system, suggesting that signaling through
PI 3-kinases is involved in the NA-mediated transcriptional
upregulation of hsp70 in mollusc immune cells. Exposure of
oyster hemocytes to PD098059, a MAP kinase kinase inhibitor,
which has recently allowed researchers to demonstrate that
signaling through the MAP kinase cascade is involved in key
cellular processes in bivalves (Katsu et al., 1999; Canesi et al.,
2000), had no significant effect at concentrations ≤50 µM. We
conclude that α-adrenergic induction of the hsp70 gene
promoter does not involve the MAP kinase cascade.
Interestingly, exposure to PD098059 at a concentration of 100
µM resulted in enhanced the hsp70 gene promoter induction.
Previous studies have reported that MAP kinases constitutively
repress transcriptional activation of the hsp70 gene promoter
in mammalian cells by phosphorylating serine residues within
the HSF sequence (Chu et al., 1996). Our results suggest that
a similar mechanism may exist in mollusc immune cells.
Questions finally arise concerning the significance, use and
function of hsp70 gene promoter induction via α-adrenergic
signaling in mollusc immunocytes. In an initial attempt to
answer such questions, we tested the hypothesis that αadrenergic signaling may potentiate the hsp response induced
by heat-shock in mollusc hemocytes. Our results show that
both heat-stimulated induction of the hsp70 gene promoter
and thermotolerance were higher in NA- or PE-pretreated
hemocytes (Fig. 7). Considering that NA release is an
immediate neuroendocrine response to stress in oysters
(Lacoste et al., 2001a), α-adrenergic-mediated transcriptional
upregulation of hsp70 may couple physiological stressresponses to hemocyte hsp-responses to ensure that immune
defenses are maintained under conditions of stress.
In a separate study, we found that NA leads to apoptosis
through β-adrenergic signaling in oyster immunocytes (Lacoste
et al., 2002). This result appears difficult to conciliate with the
present data showing that α-adrenergic stimulation by NA leads
to increased resistance in hemocytes and transcriptional
upregulation of hsp70, which is thought to inhibit the apoptotic
process (Samali and Orrenius, 1998). Several studies have,
however, reported that certain signaling molecules such as
reactive oxygen species, ceramides and several hormonal
messengers have the ability to induce both hsp responses and
apoptosis (Colombel et al., 1992; Samali and Orrenius, 1998).
In the light of this information, several hypotheses can be
proposed to interpret the apparent paradox between α- and βadrenergic regulations in mollusc immune cells. First, yetunidentified hemocyte subpopulations may predominantly
express α-adrenoceptors whereas others express βadrenoceptors. Both populations may thus be submitted to
different NA-mediated regulatory mechanisms. Second, both αand β-adrenergic signaling pathways may be present in the same
cells but not at the same time. For example, the presence of
certain growth factors or cytokines in the microenvironment of
hemocytes may modulate adrenoceptor expression or adrenergic
signaling and alter NA-mediated regulation, as in mammalian
lymphocytes (Genaro et al., 1993; Cazaux et al., 1995;
Cremaschi et al., 2000). Alternatively, the type of adrenoceptor
expressed by hemocytes may depend on their age. Apoptosis is
known to be necessary for the elimination of aged immune cells
Noradrenaline and hsp70 expression in mollusc immune cells
in vertebrates (Goldstein et al., 1991); the expression of βadrenoceptors may thus increase in ageing hemocytes to
facilitate their elimination through apoptotic processes.
We sincerely thank T. P. Yoshino for the constructs bearing the
Bomphalaria glabrata hsp70 gene promoter. We are also grateful to
J. Morales, S. Boulben and M. Knockaert for assistance and advice
concerning immunoblot assays. This work was supported by grants
from the Conseil Régional de Bretagne, Départements du Finistère,
Côtes d’Armor et Ille-et-Vilaine and the Section Régionale
Conchylicole de Bretagne Nord.
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