Noradrenaline and α-adrenergic signaling induce the hsp70 gene promoter in mollusc immune cells

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 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 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.

NA, the α-adrenoceptor agonist phenylephrine (PE), the β-adrenoceptor 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.

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).

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 reporter-gene 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.

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.

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-(2-aminoethyl)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 Tris-HCl, 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.

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.

Data are presented as means and standard errors of at least three 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.

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 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 α-adrenoceptor 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 expressed. These results suggest that NA acts through α-adrenoceptors 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 ε1 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 β1-isozymes in mammals, also blocked the activation of luciferase 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-adrenoceptor-mediated 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 ε1 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 PE-treated 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 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 α-adrenergic stimulation leads to higher thermotolerance in these cells.

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 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 that they are of relevance in vivo. As a consequence, NA-mediated 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 α-adrenoceptors (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 PTX-sensitive 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-adrenoceptor-stimulated responses are predominantly mediated by PTX-insensitive G-proteins, likely the Gq family. However, it is coherent with other studies showing that PTX-sensitive G-proteins 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 β-adrenoceptor/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 PTX-sensitive 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, α-adrenoceptors 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 α-adrenergic 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 stress-responses 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 β-adrenergic regulations in mollusc immune cells. First, yet-unidentified hemocyte subpopulations may predominantly express α-adrenoceptors whereas others express β-adrenoceptors. Both populations may thus be submitted to different NA-mediated regulatory mechanisms. Second, both α- and 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 in vertebrates (Goldstein et al., 1991); the expression of β-adrenoceptors 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.