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

Station Biologique de Roscoff, CNRS – Université Paris VI – INSU, Place Georges Teissier, BP 74, F-29682 Roscoff Cedex, France

View larger version (16K): [in a new window]   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.

View larger version (26K): [in a new window]   Fig. 2. -Adrenergic stimulation but not ß-adrenergic stimulation induces the hsp70 gene promoter in oyster immune cells. Luciferase activity increased in transfected cells exposed to the -adrenoceptor agonist PE (A), but not in cells exposed to the ß-adrenoceptor agonist isoproterenol (B). Pretreatment with the -adrenoceptor antagonist prazosin blocked the NA-induced increase in luciferase activity (C), whereas pretreatment with the ß-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.

View larger version (6K): [in a new window]   Fig. 3. Noradrenaline and the -adrenoceptor agonist PE induce expression of the inducible hsp70 isoform (lower band) in oyster immune cells, whereas in cells exposed to the ß-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).

View larger version (33K): [in a new window]   Fig. 4. -Adrenergic induction of the hsp70 gene involves a PTX-sensitive 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 PE-induced 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.

View larger version (15K): [in a new window]   Fig. 5. -Adrenergic induction of the hsp70 gene involves a PI 3-kinase. Pretreatment of transfected oyster hemocytes with the PI 3-kinase 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.

View larger version (24K): [in a new window]   Fig. 6. -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.

View larger version (18K): [in a new window]   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).

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