Glucocorticoid-mediated attenuation of the hsp70 response in trout hepatocytes involves the proteasome

The physiological implication of elevated cortisol levels on cellular heat-shock protein 70 (hsp70) response was examined using primary cultures of rainbow trout (Oncorhynchus mykiss) hepatocytes. Trout hepatocytes treated with cortisol, the predominant glucocorticoid in teleosts, responded to the heat shock (+15 degrees C for 1 h) with a significant drop in hsp70 accumulation over a 24-h recovery period. [(35)S]methionine incorporation and pulse-chase studies confirmed that this cortisol impact was due to decreased hsp70 synthesis and not enhanced protein breakdown. Cortisol also significantly decreased glucocorticoid receptor (GR) expression in trout hepatocytes. This receptor downregulation was inhibited by the proteasomal inhibitors, lactacystin and MG-132, implying a role for the proteasome in GR downregulation by cortisol. Inhibiting the proteasome did not significantly modify heat-induced hsp70 accumulation in the absence of cortisol but significantly elevated hsp70 expression in the presence of cortisol in heat-shocked trout hepatocytes. Taken together, our results suggest proteasome-mediated GR degradation as a mechanism for the attenuation of hsp70 response by cortisol in heat-shocked hepatocytes.     

Differences in metallothionein gene expression in primary cultures of rainbow trout hepatocytes and the RTH-149 cell line

Primary cultures of rainbow trout, Salmo gairdneri, hepatocytes were used to study the expression of metallothionein (MT) genes in response to steroid hormone treatment. The expression pattern was compared to that of an immortal cell line (RTH-149). MT mRNA accumulated in both cell cultures after exposure to zinc while 17 beta-oestradiol had no effect in either system. Treatment with cortisol and corticosterone resulted in a 2-fold increase of metallothionein mRNA levels in the primary cultures but had no effect in the RTH-149 cell culture. Primary cultures that were exposed to zinc or cortisol showed a high temporal correlation (r = 0.974) between MT mRNA and MT protein levels. The basal level expression was 3-4-fold higher in primary cultures than in RTH-149 cells. The present study demonstrates the inducibility of rainbow trout MT genes in response to glucocorticoids. It further indicates that primary cultures are to be preferred to immortal cell lines when investigating the inducibility of MT mRNA.     

A comparison of cadmium-induced metallothionein gene expression and Me2+ distribution in the tissues of cadmiumsensitive (rainbow trout; Salmo gairdneri) and tolerant (stone loach; Noemacheilus barbatulus) species of freshwater fish

1. The accumulation of cadmium in the liver, kidney and gills of rainbow trout and stone loach was measured during exposure of the fish to the metal at 3 smg/l in their aquarium water. The pattern of accumulation of the toxic metal in the individual organs was different between the two species.2. The tissue concentrations of metallothionein-specific mRNA and metallothionein protein were also determined in these organs from the same fish. In rainbow trout, the induction of metallothionein gene expression resulted in a gradual increase in metallothionein concentration in gill over the course of the experiment whereas increases in metallothionein in the liver and kidney were detected only at the later time points of analysis (beyond 19 weeks). By contrast, in the same tissues from stone loach, relatively minor changes were quantified in specific mRNA and metallothionein concentrations.3. Throughout the experimental period, tissue concentrations of zinc and copper were determined in the liver, kidney and gills of the rainbow trout and stone loach. Subtle decreases were observed in the zinc concentration of gills in rainbow trout and substantial increases were observed in the hepatic copper concentrations in both species at the later time points of analysis.4. The ability of cadmium to induce metallothionein gene expression and its subsequent ability to compete for the sequestration sites on the newly-synthesized protein is discussed with regard to the relative levels of cadmium, zinc and copper in the organs studied and differing regimes of cadmium administration.     

Temporal changes in stress and tissue-specific metabolic responses to municipal wastewater effluent exposure in rainbow trout

Sub-chronic exposure to municipal wastewater effluent (MWWE) in situ was recently shown to impact the acute response to a secondary stressor in rainbow trout (Oncorhynchus mykiss). However, little is known about whether MWWE exposure in itself is stressful to the animal. To address this, we carried out a laboratory study to examine the organismal and cellular stress responses and tissue-specific metabolic capacity in trout exposed to MWWE. Juvenile rainbow trout were exposed to 0, 20 and 90% MWWE (from a tertiary wastewater treatment plant), that was replenished every 2d, for 14 d. Fish were sampled 2, 8 or 14 d post-exposure. Plasma cortisol, glucose and lactate levels were measured as indicators of organismal stress response, while inducible heat shock protein 70 (hsp70), constitutive heat shock protein 70 (hsc70) and hsp90 expression in the liver were used as markers of cellular stress response. Impact of MWWE on cortisol signaling was ascertained by determining glucocorticoid receptor protein (GR) expression in the liver, brain and, heart, and metabolic capacity was evaluated by measuring liver glycogen content and tissue-specific activities of key enzymes in intermediary metabolism. Plasma glucose and lactate levels were unaffected by exposure to MWWEs, whereas cortisol showed a transient increase in the 20% group at 8d. Liver hsc70 and hsp90, but not hsp70 expression, were higher in the 90% MWWE group after 8d. There was a temporal change in GR expression in the liver and heart, but not brain of trout exposed to MWWE. Liver glycogen content and activities liver gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK), and alanine aminotransferase (AlaAT) were significantly affected by MWWE exposure. The glycolytic enzymes pyruvate kinase (PK) and hexokinase (HK) activities were significantly higher temporally by MWWE exposure in the gill and heart, but not in the liver and brain. Overall, a 14 d exposure to MWWE evokes a cellular stress response and perturbs the cortisol stress response in rainbow trout. The tissue-specific temporal changes in the metabolic capacity suggest enhanced energy demand in fish exposed to MWWE, which may eventually lead to reduced fitness.     

Decreased glucocorticoid receptor activity following glucocorticoid receptor antisense RNA gene fragment transfection.

Depression is often characterized by increased cortisol secretion caused by hyperactivity of the hypothalamic-pituitary-adrenal axis and by nonsuppression of cortisol secretion following dexamethasone administration. This hyperactivity of the hypothalamic-pituitary-adrenal axis could result from a reduced glucocorticoid receptor (GR) activity in neurons involved in its control. To investigate the effect of reduced neuronal GR levels, we have blocked cellular GR mRNA processing and/or translation by introduction of a complementary GR antisense RNA strand. Two cell lines were transfected with a reporter plasmid carrying the chloramphenicol acetyltransferase (CAT) gene under control of the mouse mammary tumor virus long terminal repeat (a glucocorticoid-inducible promoter). This gene construction permitted assay of the sensitivity of the cells to glucocorticoid hormones. Cells were also cotransfected with a plasmid containing 1,815 bp of GR cDNA inserted in the reverse orientation downstream from either a neurofilament gene promoter element or the Rous sarcoma virus promoter element. Northern (RNA) blot analysis demonstrated formation of GR antisense RNA strands. Measurement of the sensitivity of CAT activity to exogeneous dexamethasone showed that although dexamethasone increased CAT activity by as much as 13-fold in control incubations, expression of GR antisense RNA caused a 2- to 4-fold decrease in the CAT response to dexamethasone. Stable transfectants bearing the GR antisense gene fragment construction demonstrated a 50 to 70% decrease of functional GR levels compared with normal cells, as evidenced by a ligand-binding assay with the type II glucocorticoid receptor-specific ligand [3H]RU 28362. These results validate the use of antisense RNA to GR to decrease cellular response to glucocorticoids.     

Increased abundance of labile intracellular zinc during cell proliferation was due to increased retention of extracellular zinc in 3T3 cells

Platelet-derived growth factor (PDGF)-, epidermal growth factor (EGF)- and insulin-like growth factor I (IGF-I)-stimulated cell proliferation in 3T3 cells was accompanied by increased abundance of labile intracellular pool of zinc (LIPZ). However, the origin and regulation of this cell proliferation-associated increase in the abundance of LIPZ are unknown. Cellular zinc homeostasis involves zinc transporters and metallothionein. The objectives of this study were to determine whether cell proliferation-associated increase in the abundance of LIPZ was a result of an increased zinc uptake and to assess the involvement of zinc transporters and metallothionein in this cell proliferation-associated increase in the abundance of LIPZ in 3T3 fibroblasts. Zinc transporters assessed included both zinc importer (Zip1) and zinc exporters (ZnT1, ZnT2 and ZnT4). Growth factors increased the abundance of LIPZ while total cellular zinc concentration remained unaffected, demonstrating that LIPZ was responsive to the increased needs for zinc during growth factor-stimulated cell proliferation. Growth factors also increased net zinc retention as indicated by higher 65zinc radioactivity and elevated mRNA levels of Zip1, ZnT1 and ZnT4. Although zinc is essential to cell proliferation, excessive cellular zinc accumulation causes cytotoxicity. Collectively, these observations suggest that increase in the abundance of LIPZ during growth factor-stimulated cell proliferation was due to increased net retention of extracellular zinc, which was apparently achieved through a coordinated up-regulation of the expression of transporters involved in both zinc influx and efflux to ensure adequate supply of zinc to sustain cell proliferation, yet to prevent potential zinc cytotoxicity in 3T3 cells.     

Glucocorticoid receptor immunoreactivity in neurons and pituitary cells implicated in reproductive functions in rainbow trout: a double immunohistochemical study

In order to identify the nature of the glucocorticoid receptor (GR)-expressing neurons and pituitary cells that potentially mediate the negative effects of stress on reproductive performance, double immunohistochemical stainings were performed in the brain and pituitary of the rainbow trout (Oncorhynchus mykiss). To avoid possible cross-reactions during the double staining studies, combinations of primary antibodies raised in different species were used, and we report here the generation of an antibody raised in guinea pig against the rainbow trout glucocorticoid receptor (rtGR). The results obtained in vitellogenic females showed that GnRH-positive neurons in the caudal telencephalon/anterior preoptic region consistently exhibited rtGR immunoreactivity. Similarly, in the anterior ventral preoptic region, a group of tyrosine hydroxylase-positive neurons, known for inhibiting gonadotropin (GTH)-2 secretion during vitellogenesis, was consistently shown to strongly express GR. Finally, we show that a large majority of the GTH-1 (FSH-like) and GTH-2 (LH-like) cells of the pituitary exhibit rtGR immunoreactivity. These results indicate that cortisol may affect the neuroendocrine control of the reproductive process of the rainbow trout at multiple sites.     

虹鳟鱼金属硫蛋白启动子的体外扩增、克隆及其序列分析

以含有虹鳟鱼金属硫蛋白基因的质粒ＤＮＡ为模板,以合成的两段３２个寡聚核苷酸为引物,经过ＰＣＲ扩增,合成了４３３ｂｐ的片段,把其克隆到ｐＢＬ－ＣＡＴ载体中,序列分析和限制酶切图谱表明所克隆的虹鳟鱼金属硫蛋白启动子含有４３３ｂｐ,含有ＴＡＴＡ和ＣＡＡＴ序列,与Ｈｏｎｇ报导的鳟鱼金属硫蛋白启动子的序列比较,其核苷酸泊同源率为１００％。     

Induction of metallothionein gene expression by cadmium and the retention of the toxic metal in the tissues of rainbow trout (Salmo gairdneri)

1. When rainbow trout were exposed to cadmium by intraperitoneal injection, there was a rapid (within 3hr) and significant (approx. 63%) loss of the metal from the whole bodies of the fish.2. Of the metal retained in the bodies of the fish (approx. 37% of the injected dose), more than 98% was accounted for collectively among the liver, kidney and gills.3. Subsequent maintenance of the rainbow trout in fresh water for up to 98 days post-metal administration, indicated that there was no further loss of the cadmium accumulated in the organs studied and that the distribution of the metal among the liver, kidney and gills remained unchanged over that period.4. During this 98-day period of maintenance of the fish, tissue concentrations of metallothionein-specific mRNA and metallothionein protein were quantified using riboprobe and ELISA systems respectively. Metallothionein-specific mRNA concentrations increased rapidly (within 24 hr) before falling back to levels similar to, or slightly greater than, those found in control animals. The concentration of metallothionein protein also increased significantly (within 3 days) then remained elevated thereafter.5. Throughout the experimental period, the concentrations of zinc and copper were also monitored in the liver, kidney and gills of the rainbow trout. The concentrations of each ion differed between each of the organs but did not change during the experiment.6. The induction of metallothionein gene expression by cadmium in the liver, kidney and gill of rainbow trout and the subsequent sequestration of the toxic metal is discussed with regard to the relative levels of these other essential metal ions.     

Endogenous and heavy-metal-ion-induced metallothionein gene expression in salmonid tissues and cell lines

Endogenous levels of metallothionein (MT) mRNA were detected by RNA probes in several somatic and germ-line tissues of rainbow trout, such as eggs, ovaries and immature testis. These levels may be related to metal-ion homeostasis in the observed tissues. The induction kinetics of trout MT isoform B (MT-B) mRNA were studied after single intraperitoneal injections of CdCl2, CuCl2 and ZnCl2. MT-B mRNA was induced within 12 h in liver, kidney, spleen and gills. However, over the 48-h experimental period, the kinetics of MT-B mRNA accumulation differed in response to the three metal salts, possibly due to differential handling of the salts by these tissues. Multiple metal-salt injections induced high levels of MT-B mRNA in the four tissues studied. In the rainbow trout hepatoma cell line, ZnCl2 was a better inducer of the MT-B gene, as compared to CdCl2 and CuCl2. The expression of the exogenous trout MT-B promoter in Chinook salmon embryonic cell line indicates the presence of MT regulatory factors. In contrast, the endogenous MT genes in these cells are quiescent, possibly due to the methylation of their promoter region.     

The expression of a synthetic rainbow trout metallothionein gene in E. coli

A synthetic gene for rainbow trout metallothionein was constructed and inserted into a dual origin plasmid where expression was induced by a temperature shift in a proteinase-deficient strain of Escherichia coli. The recombinant protein was purified to homogeneity, and a partial amino acid sequence was determined to confirm its identity. Its immunochemical characteristics were similar to those of native metallothionein from rainbow trout. The amounts of recombinant metallothionein produced were quantified in soluble cell extracts by ELISA. Low concentrations were detected when growth was performed either in L-broth or defined (GMM-II) medium. Supplementation of the medium with zinc or copper had no effect on the amount of metallothionein produced. By contrast, when cadmium was included in either L-broth or GMM-II medium, much higher concentrations of the protein within the cells (approx. 13 micrograms/mg soluble cell protein) were detected. This stabilisation of the protein by metal reconstitution in vivo is considered in relation to the selective uptake/exclusion of metals by the cells and its significance for the scavenging of certain precious or toxic heavy metals is discussed.     

Cortisol receptor expression differs in the brains of rainbow trout selected for divergent cortisol responses

In rainbow trout (Oncorhynchus mykiss), selection for divergent post-stress plasma cortisol levels has yielded low (LR)- and high (HR) responsive lines, differing in behavioural and physiological aspects of stress coping. For instance, LR fish display prolonged retention of a fear response and of previously learnt routines, compared to HR fish. This study aims at investigating putative central nervous system mechanisms controlling behaviour and memory retention. The stress hormone cortisol is known to affect several aspects of cognition, including memory retention. Cortisol acts through glucocorticoid receptors 1 and 2 (GR1 and 2) and a mineralcorticoid receptor (MR), all of which are abundantly expressed in the salmonid brain. We hypothesized that different expressions of MR and GRs in LR and HR trout brains could be involved in the observed differences in cognition. We quantified the mRNA expression of GR1, GR2 and MR in different brain regions of stressed and non-stressed LR and HR trout. The expression of MR was higher in LR than in HR fish in all brain parts investigated. This could be associated with reduced anxiety and enhanced memory retention in LR fish. MR and GR1 expression was also subject to negative regulation by stress in a site-specific manner.