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Am J Physiol Regul Integr Comp Physiol 294: R623-R629, 2008. First published December 12, 2007; doi:10.1152/ajpregu.00646.2007
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COMPARATIVE AND EVOLUTIONARY PHYSIOLOGY

Cortisol stimulates the zinc signaling pathway and expression of metallothioneins and ZnT1 in rainbow trout gill epithelial cells

Nic R. Bury, Mi Ja Chung, Armin Sturm, Paul A. Walker, and Christer Hogstrand

Nutritional Sciences Division, King's College, London, United Kingdom

Submitted 7 September 2007 ; accepted in final form 10 December 2007


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
Intracellular zinc signaling is important in the control of a number of cellular processes. Hormonal factors that regulate cellular zinc influx and initiate zinc signals are poorly understood. The present study investigates the possibility for cross talk between the glucocorticoid and zinc signaling pathways in cultured rainbow trout gill epithelial cells. The rainbow trout metallothionein A (MTA) gene possesses a putative glucocorticoid response element and multiple metal response elements 1042 base pairs upstream of the start codon, whereas metallothionein B (MTB) and zinc transporter-1 (ZnT1) have multiple metal response elements but no glucocorticoid response elements in this region. Cortisol increased MTA, MTB, and ZnT1 gene expression, and this stimulation was enhanced if cells were treated with cortisol together with zinc. Cells treated with zinc showed increased zinc accumulation, transepithelial zinc influx (apical to basolateral), and intracellular labile zinc concentrations. These responses were also significantly enhanced in cells pretreated with cortisol and zinc. The cortisol-mediated effects were blocked by the glucocorticoid receptor (GR) antagonist RU-486, indicating mediation via a GR. In reporter gene assays, zinc stimulated MTA promoter activity, whereas cortisol did not. Furthermore, cortisol significantly reduced zinc-stimulated MTA promoter activity in cells expressing exogenous rainbow trout GR. These results demonstrate that cortisol enhances cellular zinc uptake, which in turn stimulates expression of MTA, MTB, and ZnT1 genes.

glucocorticoid receptor; metal-regulatory transcription factor-1; fish; metals; glucocorticoid response element

METALLOTHIONEINS (MT) are small cysteine-rich metal-binding proteins that in mammals exist as four major isoforms: MT-I, -II, -III, and -IV (53). MT-I and MT-II constitute metal-inducible intracellular proteins that are expressed in most cells. In contrast, MT-III and MT-IV are not metal inducible and are expressed specifically in the central nervous system and squamous epithelia, respectively (40, 51). The biological functions of MT-I and MT-II include buffering of intracellular zinc, protection against metal toxicity (38, 43), and defense against oxidative stress (6, 12). The MTs found in different fish species are homologous to the mammalian MT and are functionally equivalent to the MT-I and MT-II isoforms (31).

The induction of MT expression by zinc is coordinated by metal-regulatory transcription factor-1 (MTF1; Ref. 17). MTF1 is evolutionary conserved, and orthologues are present in Drosophila melanogaster, fish, mice, and humans (3, 8, 15, 56). Zinc causes MTF1 to translocate to the nucleus (47, 48) and enhances MTF1 binding to metal response elements (MRE) of target genes, including MT (2). Similar to the situation in mammals, MT genes in fish and many other species characteristically possess multiple MRE that drive MT transcription in response to MTF1 binding (7, 31, 37, 41). MTF1 also enhances expression of a growing list of genes involved in zinc homeostasis and responses to metal toxicity and oxidative stress (1, 47). A central theme to the mechanism of MT induction appears to be the ability of MTF1 to act as a sensor for labile intracellular zinc (2).

Transcription factors other than MTF1 are known to contribute to MT promoter activation in various species. There is conclusive evidence that glucocorticoids can induce MT expression in humans and mice independent of other gene activation factors (15, 28, 29). The response to glucocorticoids on MT expression in fish, on the other hand, is controversial. In vivo studies have shown that different types of stress (confinement, salinity exposure) can induce expression of MT or MT-like proteins in the liver of various fish species (11, 34, 44). Furthermore, treatment of cultured primary rainbow trout hepatocyte cultures with a supraphysiological concentration of cortisol (10 µM) has been shown to stimulate MT expression (36). In contrast, MT expression in the rainbow trout hepatoma (RTH-149) cells is unresponsive to cortisol treatment (36). Similarly, HepG2 and HEK-293 cells transfected with a reporter gene driven by the crucian carp MT promoter, which has two consensus glucocorticoid response elements (GREs), were unaffected by the synthetic corticosteroid dexamethasone (41). Given the equivocal data on cortisol-mediated MT expression in teleost fish, we hypothesized that cortisol-induced MT expression may not be directly regulated via glucocorticoid receptor (GR) binding to the GRE in the promoter region of the MT gene but via interaction between the glucocorticoid and zinc signaling pathways. In this scenario, cortisol would cause an increase in the intracellular concentration of labile zinc, triggering MT expression via the classical MTF1/MRE pathway.

In rainbow trout, there are two MT isoforms, MTA and MTB (7), which both possess tandem MREs located within 1–100 bp of the transcription initiation site and which are especially important for metal inducibility of the genes (33, 37, 45, 46). Rainbow trout MTA and MTB also have distal MRE clusters (less than –500 bp), and these are also characteristic of teleost fish MT promoters (21, 31, 41, 50). However, there are also several differing cis-acting elements in their 5'-flanking regions (Refs. 37, 45; Fig. 1); importantly for this study, the MTA promoter region possesses a GRE, whereas that of MTB does not. Thus cortisol may be expected to induce expression of MTA but not of MTB. In contrast, if cortisol stimulates MT expression via the zinc signaling pathway, then the expression of both MT isoforms will be expected to increase, as well as the expression of other MTF1/MRE-driven genes. Consequently, in addition to MTA and MTB, we measured expression of the gene encoding for solute carrier 30a1 (slc30a1), also known as zinc transporter-1 (ZnT1) (4), whose –2 kb of the 5'-flanking region in torafugu, zebrafish, and humans has no GRE motifs but several MREs (4).


Figure 1
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  		Fig. 1. Metallothionein A (MTA; top) and metallothionein B (MTB; bottom) promoter regions identified by Olsson and colleagues (37) and Samson and Gedamu (45). Putative transcription binding sites are shown for IL-6 (NF-IL-6), glucocorticoid response element (GRE), antioxidant response element (ARE), activation protein-1 (AP1), and metal-regulatory transcription factor-1 [MTF1; a metal response element (MRE)].

 

   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
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Chemicals. FBS, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenol tetrazolium bromide (MTT), NaCl, KCl, Na2HPO4, KH2PO4, ZnSO4·7H2O, HEPES, EDTA, hydrocortisone, mifepristone (RU-486), DMEM nutrient mixture F-12 Ham-Bradford reagent, and TriReagent LS were purchased from Sigma-Aldrich. DMEM was purchased from Invitrogen. Leibovitz (L-15) medium with L-glutamine and phenol red, penicillin, and streptomycin, fungizone reagent, gentamycin reagent solution liquid, and trypsin-EDTA were obtained from GIBCO BRL. Cell strainers (100 µm; Falcon), 0.4-µm cell inserts (12 well; Falcon), tissue culture insert companions (12 well; Falcon), and tissue culture plate (6 well) were purchased from Marathon. Powerscript reverse transcriptase and the plasmid pSVβ were obtained from Clonetech. RNase-free glycogen, DNase I, oligo(dT)15 primer and random hexamers, luciferase assay kit, and the pGL2-Basic plasmid were sourced from Promega. Gene-specific probes were from Qiagen. Pico green and FluoZin-3 were purchased from Molecular Probes. 65Zn was obtained from Perkin-Elmer.

Gill cell culture. Rainbow trout (Oncorhynchus mykiss) were euthanized, and the gill cells were isolated according to the procedure described by Kelly et al. (30) and modified by Walker et al. (54) to improve functionality and metal responsiveness. The L-15 medium on the apical side of the epithelium was replaced with 1.5 ml of synthetic moderately hard water (1.14 mM NaHCO3, 0.35 mM CaSO4, 0.5 mM MgSO4, 54.1 µM KCl; pH 7.6) containing 0, 5, or 10 µM of ZnSO4 (all zinc concentrations are nominal). These zinc levels are within the environmental range and correspond approximately to 0, 35, and 70% of the 96-h LC50 to rainbow trout in similar water hardness (54). The L-15 medium, which contains no zinc, in the basolateral compartment was replaced with fresh L-15 medium, which was supplemented with 0.25 or 1.0 µM cortisol and in some experiments in combination with 1 µM of the GR inhibitor RU-486. After the different treatments for 24 h, cell culture inserts were bathed in 0.75 ml of TriReagent LS, the RNA was isolated, and first-strand cDNA was produced with Powerscript reverse transcriptase with oligo(dT)15 primer and random hexamers, following the manufacturer's guidelines. Quantitative real-time PCR amplification was carried out on an ABI Prism 7000 sequence detection system (Applied Biosystems) using the following specific rainbow trout primers: 18S rRNA (GenBank accession no. AF-157514), with forward primer 5'-cggaggttcgaagacgatca-3' and reverse primer 5'-tcgctagttggcatcgtttatg-3'; MTA (M18103 [GenBank] ), with forward primer 5'-catgcaccagttgtaagaaagca-3' and reverse primer 5'-gcagcctgaggcacacttg-3'; MTB (M18104 [GenBank] ), with forward primer 5'-tcaacagtgaaattaagctgaaatacttc-3' and reverse primer 5'-aagagccagttttagagcattcaca-3; and ZnT1 (AY-742790), with forward primer 5'-acaacagcccccctaatgg-3' and reverse primer 5'-gttcatctgcatctcactgtcgtt-3'. The gene-specific probes used were 5'-JOE ataccgtcgtagttccg BHQ-3' for 18S rRNA, 5'-VIC tgctgcgactgctgt MGB-3' for MTA, 5'-6-FAM tttgactaaagaagcg BHQ-3' for MTB, and 5'-6-FAM tgaacaccgagagacgta BHQ-3' for ZnT1 in a buffer containing 200 mM Tris·HCl (pH 8.3), 500 mM KCl, 40 mM MgCl2, and 10 µM ROX. All reactions were carried out in triplicate.

Intracellular chelatable Zn2+. Relative concentrations of intracellular chelatable Zn2+ were measured in gill cells cultured on six-well plates. Cultured gill epithelia were pretreated with 1 µM cortisol, 100 µM ZnSO4, 1 µM RU-486, or combinations for 24 h. Cells kept in L-15 only served as controls. The cells were washed three times with PBS and then incubated with 3 µM of the zinc-specific probe FluoZin-3-AM in L-15 medium. After a 60-min incubation, the cells were washed three times with L-15 and then incubated for another 30 min in L-15 medium to complete the intracellular cleavage of the FluoZin-3-AM. One milliliter of 135 mM NaCl, 1.1 mM EGTA, and 20 mM HEPES, pH 7.5, was added, and the cells were scraped off the inserts and homogenized. The homogenate (50 µl) was diluted with 2.0 ml of the same buffer, and fluorescence was measured with a spectrofluorophotometer (Perkin-Elmer, LS 50B) with excitation at 494 nm and emission at 516 nm. Intensity of fluorescence was normalized to protein concentration, as measured with Bradford reagent, and chelatable zinc concentrations in treated gill epithelia were expressed as percentage of the control.

Zinc transport experiments. Unidirectional influx of zinc across the cultured gill epithelium and rate of zinc accumulation into the cells was measured radioisotopically by 65Zn. Cultured gill epithelia were pretreated for 20 h with L-15 only or medium with 1 µM cortisol (basolateral compartment), 100 µM ZnSO4 (apical compartment), or 1 µM cortisol (basolateral compartment) in combination with 100 µM ZnSO4 (apical compartment). The medium on the basolateral side was then replaced with 2.0 ml of fresh L-15 without additives, and the apical medium was replaced with 1.5 ml of synthetic moderately hard water, containing 100 µM ZnSO4 and 1.5 kBq/ml of 65Zn. Flux of zinc was allowed to progress for 40 min, after which 1.0-ml aliquots of the media on either side of the epithelium were withdrawn for radioactivity counting (LKB Wallac, 1282 Compugamma). There was no change in volumes of media on the two sides of the cultured gill cells during the 40-min flux experiment. The cells were finally lysed with 1.7 ml of 1 M NaOH, of which 5 µl were saved for protein analysis; the remaining lysate was counted for radioactivity to determine cellular accumulation of zinc. Unidirectional influx of zinc across the epithelium was calculated from the total 65Zn radioactivity on the basolateral side and the specific activity of 65Zn (Bq/mol) in the apical compartment. Similarly, zinc accumulation rate was calculated from the total amount of radioactivity in the cells and normalized to protein content and the specific activity of 65Zn in the apical compartment.

Expression vector constructs. The expression plasmid pCMrtGR1 (previously called pCMrtGR) encoding rainbow trout corticosteroid receptor rtGR1 has been described elsewhere (16). The reporter plasmid pGL2MTA-Luc, in which luciferase expression is driven by the rainbow trout MTA promoter, was obtained as described by Olsson et al. (37). The plasmid pSVβ, in which galactosidase expression is under the control of the SV40 promoter, was used to control transfection efficiency, whereas pBluescript SK(+) (X52325 [GenBank] ) was used as an irrelevant plasmid to top up DNA amounts.

Reporter gene transactivation assays. COS-7 cell culture and tranfection protocol followed that described in Sturm et al. (52). Cells cultured in 12-well plates were transfected with 5 µg of pGL2MTA-Luc, 2 µg of pSVβ, 10 µg of pVBluescript SK(+), and up to 5 µg of pCMrtGR1. If >5 µg of pCMrtGR1 were used, empty vector pCMV5 (AF-239249) was added to give the same total DNA concentration. Twelve hours after transfection, cells were washed with PBS, the medium was renewed, and cortisol (1 µM) and/or zinc sulfate (80 µM) was added from 1,000-fold concentrated stock solutions, which had been prepared in ethanol for cortisol and in sterile water for zinc sulfate. After 30 h of incubation, cell extracts were analyzed for luciferase and β-galactosidase activities (22). If the reporter plasmid pGL2MTA-Luc was replaced by empty vector pGL2-Basic, luciferase activities was virtually absent in transfected cells regardless of treatment (data not shown). Experiments were repeated at least three times independently, with triplicate cell cultures per treatment. Luciferase activity was corrected for well-specific transfection efficiency as determined by β-galactosidase activity.

Statistical analysis. Data are expressed as means ± SD and are average values from three to nine values per experiment; experiments were repeated at least twice to confirm results. One-way ANOVA followed by Tukey's test was used to compare the results from different treatments. For the transfection assays, results are expressed as a percentage of zinc-stimulated control (e.g., cells transfect with the pGL2MTA-Luc plasmid only and treated with zinc). Percentages were log transformed and assessed via a paired t-test. Statistical significance level was set to P < 0.05.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
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DISCUSSION
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MTA, MTB, and ZnT1 gene expression in primary gill cell culture. A primary gill cell culture grown on semi-permeable supports was used to assess the specific effects of cortisol and zinc on MTA, MTB, and ZnT1 gene expression in rainbow trout. This gill cell culture system retains transepithelial resistance and polarity characteristics of the intact organ, enabling water to be placed on the apical side while the basolateral surface is bathed in L-15 medium (30, 54). When 5 µM zinc was added to the apical compartment, MTA and ZnT1 mRNA but not MTB mRNA were significantly increased compared with untreated cells (Fig. 2). With addition of 10 µM waterborne zinc, expression of all three genes was elevated compared with gene expression levels in control cells. Cortisol (applied basolaterally) induced expression of MTA, MTB, and ZnT1, but the genes showed different sensitivities to the hormone treatment. When 1 µM cortisol was added, resulting in plasma levels being equivalent to those recorded during severe stressful stimuli (55), all three genes displayed significantly increased expression above the untreated control (Fig. 2). With 0.25 µM cortisol, MTA and ZnT1 were significantly upregulated; with 0.1 µM cortisol, none of the genes investigated was upregulated. Combined treatment with 0.25 µM cortisol and either 5 or 10 µM waterborne zinc significantly enhanced expression of MTA, MTB, and ZnT1, at levels above those shown for untreated control and those for treatment with cortisol or zinc alone (Fig. 3). The enhanced effect of cortisol in combination with zinc on ZnT1 expression was likely mediated by the GR because it was abolished in the presence the GR blocker RU-486 (Fig. 3).


Figure 2
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  		Fig. 2. Induction of MTA (open bars), MTB (hatched bars), and zinc transporter-1 (ZnT1; cross-hatched bars) gene expression in primary gill cells treated with zinc (5 or 10 µM) or cortisol (0.1, 0.25, or 1 µM) or untreated (control) for 20 h. Results are expressed as fold induction of levels measured in untreated cells. Values are average + SE. *Significant difference from untreated cells (ANOVA followed by a Tukey-Kramer post hoc test, P < 0.05, using untransformed data).

 

Figure 3
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  		Fig. 3. Induction of MTA (top), MTB (middle), and ZnT1 (bottom) gene expression in primary gill cells treated with zinc (5 or 10 µM), cortisol (0.25 µM), or zinc (5 or 10 µM) and cortisol (0.25 µM) in combination and in the presence or absence of the glucocorticoid receptor (GR) agonist RU-486 (1 µM) for 20 h. Results are expressed as fold induction of untreated cells (control). Values are average + SE. Bars with different letters are significantly different from each other (ANOVA followed by a Tukey-Kramer post hoc test, P < 0.05, using untransformed data).

 
Zinc influx and intracellular chelatable zinc. The positive effects of cortisol on the expression of MTB and ZnT1, neither of which have obvious GRE –1000-bp and –2000-bp regions upstream of the start codon, respectively, led us to postulate that cortisol may increase intracellular zinc concentrations, in turn resulting in increased ZnT1 and MTB expression via the zinc-mediated MTF1 signaling pathway. The fluorescent zinc probe FluoZin-3 (Kd = 15 nM) was used to determine the effects of cortisol and elevated external zinc levels on relative intracellular labile zinc concentrations. The cells were treated for 24 h with cortisol (1 µM), zinc (100 µM), or a combination, as well as in the presence or absence of RU-486 (1 µM), after which intracellular concentrations of FluoZin-3 chelatable zinc were determined. Cortisol alone, without any zinc present in the medium, did not affect the chelatable zinc concentration of the gill cells. In contrast, the addition of 100 µM zinc to the culture medium significantly increased the intracellular chelatable zinc concentration by 1.6-fold above results shown for control. Furthermore, the combination of 1 µM cortisol and 100 µM zinc substantially enhanced the intracellular labile zinc concentrations above those present in cells receiving the single treatments with cortisol or zinc. Again, it would appear that the cortisol component of this response was mediated by GR because addition of 1 µM RU-486 restored the intracellular chelatable zinc concentration back to the same level as that of the treatment with zinc alone (Fig. 4). These results suggest that cortisol had a stimulatory influence on the concentration of intracellular chelatable zinc, enabling it to rise when exogenous zinc was added to the system.


Figure 4
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  		Fig. 4. Fluorescence readings, expressed as a percentage of untreated cells, of primary gill cells incubated with 3 µM FluoZin 3 for 60 min after treatment with zinc (100 µM), cortisol (1 µM), or zinc (100 µM) and cortisol (1 µM) in combination in the presence or absence of the GR agonist RU-486 (1 µM) for 20 h. Values are average + SE. Bars with a different letters are significantly different from each other (ANOVA followed by a Tukey-Kramer post hoc test, P < 0.05, using untransformed data).

 
The increase in intracellular zinc could be due to either release of zinc from intracellular stores or an increase in uptake of zinc from the extracellular compartment. To assess the latter possibility, cellular accumulation rate and transepithelial influx of zinc (from the apical to the basolateral compartment), were measured over a 3-h period by the addition of 10 µM 65Zn to the apical side and measuring the appearance of radiolabeled zinc in the cells or in the basolateral media. A 24-h pretreatment of cells with cortisol (1 µM) in the absence of zinc had no effect on either of the Zn2+ transport endpoints (Fig. 5). Pretreatment with 5 µM zinc resulted in a significant increase in transepithelial zinc influx, whereas combined treatment with zinc and cortisol further increased transepithelial zinc influx and also significantly increased the cellular zinc accumulation rate (Fig. 5).


Figure 5
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  		Fig. 5. Primary gill cell culture cellular zinc accumulation (top) and transepithelial zinc influx (bottom) after a 20-h treatment with cortisol (1 µM), zinc (5 µM), or combination of zinc and cortisol or left untreated (Con). Cellular zinc accumulation and transepithelial influx were determined by the addition of 65Zn to the apical chamber for the last 40 min of the treatment. Values are average + SE. Bars with different letters are significantly different from each other (ANOVA followed by a Tukey-Kramer post hoc test, P < 0.05, using untransformed data).

 
Reporter gene transactivation activity. To more directly investigate the effects of zinc and cortisol, individually or in combination, on MTA expression, we used a reporter gene assay system in which COS-7 cells were cotransfected with the reporter construct pGL2MTA-Luc plasmid and in which firefly luciferase expression was driven by a 1,042-kb fragment of the rainbow trout MTA 5'-promoter region and differing amounts of rtGR1 (16) expression constructs. Previously reported experiments have shown that the rainbow trout corticosteroid receptors used are functional in COS-7 cells (10). In cells transfected with pGL2MTA along with rtGR1, zinc alone (80 µM) stimulated transactivation above the medium-alone control, demonstrating the functionality of the zinc-MTF1 pathway on the reporter gene construct (Fig. 6). In contrast, cortisol alone showed no transactivational activity (Fig. 6), suggesting that the –1042-kb MTA promoter, which has a single consensus GRE, is not activated by cortisol. Increasing the concentrations of the rtGR1 plasmid had no effect on zinc-induced transactivation compared with cells transfected with pGL2MTA-Luc alone. However, at a GR1 plasmid concentration of 1.5 and 5 µg per plate, transactivational activities in the presence of zinc and cortisol were significantly reduced compared with that shown in cells transfected with pGL2MTA-Luc alone and treated with zinc (P = 0.015 and 0.013). At 5 µg GR1, treatment with zinc and cortisol significantly reduced the zinc-induced transactivational activity (P = 0.008) (Fig. 6).


Figure 6
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  		Fig. 6. MTA promoter activity in COS-7 cells without addition of zinc or cortisol (control; solid black bars) or in the presence of cortisol (1 µM; light gray bars), zinc (80 µM; open bars), or zinc (80 µM) and cortisol (1 µM) in combination (hatched bars). COS-7 cells were transiently transfected with 5 µg of pGLMTA-Luc reporter plasmid, different concentrations (0, 0.5, 1.5, and 5 µg) of rainbow trout GR expression construct, 2 µg of β-galactosidase reporter (pSVβ, to correct for transfection efficiency), and pBluescript SK to top up DNA to 12 µg. Data are expressed as a percent of the luciferase activity measured in cells transfected with only 5 µg pGLMTA-Luc and treated with 80 µM zinc (positive control). Each bar represents average + SE from 3 separate experiments. *Significant difference to the positive control, #significantly different from those cells transfected with 5 µg pGLMTA-Luc and no GR and treated with zinc and cortisol, and Asignificant difference between cells that have been transfected similarly but treated with either zinc or zinc and cortisol (paired t-test, P < 0.05 using log-transformed data).

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
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In the present study, we show that cortisol does increase MT expression in primary gill cell culture, in particular, when zinc is present in excess. Our results suggest that this stimulation is unlikely to be due to the direct effects of the cortisol-corticosteroid receptor complex acting as a ligand-inducible transcription factor and binding to the GRE present in the promoter region of the MTA gene but is a consequence of the cortisol-induced increase in the intracellular concentration of labile zinc. The elevated labile zinc activates the MTF1 signaling pathway, enhancing binding to MRE sites present in the promoter region of MT (47, 48). The hypothesis that cortisol induced MTA expression via activation of the MTF1 pathway is supported by the observation that both expression of ZnT1, a zinc exporter, and expression of trout MTB isoform gene increased in primary gill cell culture treated with cortisol. The 5'-flanking regions of ZnT1 and MTB do not possess any obvious GREs; however, similar to MTA, they are characterized by the presence of multiple MREs. The smaller effect of cortisol and zinc treatment on MTB expression compared with MTA expression may be explained by the presence of three tandem MREs in the promoter of MTA compared with one tandem MRE in the MTB promoter (36, 37, 45). However, there are two caveats to our conclusions. First, we cannot exclude the possibility of a glucocorticoid enhancer region further upstream of the MTA gene. Our transfection assay used a construct containing –1042 bp of the MTA promoter region, whereas the mouse MT1 possess two GREs separated by 6 kb (29). Second, cortisol alone stimulated expression of all three genes despite the absence of zinc in the L-15 medium and synthetic water used in our study. This could be explained by the possibility of cortisol-stimulated release of zinc from intracellular stores. Such an effect would not cause a change in FluoZin-3 readout, as this was measured on a cell homogenate. However, these caveats do not detract from the major conclusion that 1 µM cortisol stimulates zinc flux into the gill cells and raises the intracellular concentration of FluoZin-3-chelatable zinc.

Cortisol stimulates zinc uptake in a number of different cell types, including retinal pigment epithelium (35), brain (5), and HeLa (14) cells. Cortisol also affects the fast and slow exchangeable zinc pools in primary hepatocyte cultures (39). Furthermore, the retention of zinc in the perfused rat jejunum was found to be enhanced by dexamethasone administration or adrenalectomy, indicating a stimulatory effect on zinc absorbance or uptake (6). In the present study, we add to this information by showing that cortisol also increases transepithelial influx of zinc across an absorptive vertebrate epithelium, the gill, as well as the intracellular chelatable zinc concentrations.

To provide more mechanistic evidence as to whether cortisol can directly induce MTA promoter activity, a reporter plasmid (pGLMT-LUC) was constructed containing the –1042-bp 5'-flanking region of the MTA gene (37) linked to the firefly luciferase gene. Zinc treatment induced transactivation in COS-7 cells transiently transfected with the plasmid, demonstrating that the promoter was functional. In contrast, treatment with cortisol alone had no effect on MTA promoter activity in COS-7 cells cotransfected with pGLMT-LUC and plasmid expressing the recombinant rainbow trout GR1 (Ref. 16; Fig. 6). A similar result was obtained with the crucian carp MT promoter, which possesses a single GRE (41). However, the experiments with the MTA promoter revealed a curious result, in which zinc and cortisol treatment in combination appeared to reduce the MTA promoter activity in cells cotransfected with 5 µg of GR1 expression vector and the reporter plasmid (Fig. 6). The same result was obtained in cells transfected with the other rainbow trout corticosteroid receptors, rtGR2 or rtMR (10, 52), and treated in the same way (data not shown). This may suggest the presence of a negative GRE or a repressor function in this region. Indeed, a repressor of GRE function, which is responsive to stress, has been identified in the promoter region of the lactate dehydrogenase-B gene of Southern US population of killifish (Fundulus heteroclitus) (49).

Result from reporter gene experiments show that cortisol has no stimulatory effect on the –1042 bp of the MTA promoter and contradict the positive effects that cortisol and zinc treatment had on inducing genes possessing MREs in their promoter regions in primary gill cell cultures (Figs. 2 and 3). There are two potential explanations for this discrepancy. First, it is possible that cortisol is more effective in raising intracellular labile zinc concentrations in rainbow trout gill cells than in African green monkey kidney (COS-7) cells. A mechanistic basis for this is unclear but may be dependent on the differential cellular expression of zinc importers that are responsive to glucococortoids. Recently, we have reported tissue-specific responses in the transcript expression levels for various zinc transporters in zebrafish exposed to elevated or limited external zinc (17). Second, the pCMrtGR1 plasmid expression system may result in cells possessing a far greater number of GR than found in primary gill cell culture. It is not clear how GR overexpression would reduce zinc-mediated gene expression; a possibility is that GR, a protein with two zinc fingers, may compete with MTF1 for the labile zinc pool, thus acting as an intracellular zinc chelator.

Perspectives and Significance

Controversy over the use of MT as a biomarker of metal exposure in fish exists, and presently elevations in MT are considered a general stress response, rather than a metal-induced effect (43, 44). Exposure to metals causes a transient rise in tissue concentrations of metal ions (9, 19, 23) and also induces a systemic stress response in fish (55). The present study demonstrates that the rise in MT expression seen during stress is due to an increase in the intracellular labile zinc concentration, which at least partially can be explained by an enhanced apical zinc influx. The increase in metal load is probably due to the increase in expression or permeability of zinc importers in the gill cells. Therefore, dynamic shifts in membrane transporter populations during stress may greatly influence the effect that metal exposure has on that organ and in fish; cortisol-induced MT expression could be considered a metal effect. The biological significance of this mechanism needs further investigation, but it might relate to the function of zinc in cell signaling. For example, our group (12) has previously shown that zinc signaling in rainbow trout gill cells mediates expression of antioxidant proteins during oxidative stress.

The influence of GR on zinc signaling and MTF1 activation has wide implications and may be of significance for development and control of asthma, among other ailments, in which both corticosteroids and zinc have been shown to have effects. Zinc deficiency has been linked to development of asthma, and zinc supplementation has been shown to reduce the number of inflammatory eosinophil granulocytes and lymphocytes in the bronchoalveolar lavage fluid of mice with induced acute and chronic airway inflammation (32). Lang et al. (32) suggested that this reduction in inflammatory cells corresponded to a complex relationship between the expression levels of the zinc transporter, which were directed toward maintaining or increasing intracellular zinc in the lung tissue. Inhaled or orally administered glucocorticoid is the most common treatment for patients with asthma (25). Glucocorticoids suppress inflammation in the lung via the reduction in eisonophil infiltration and an increase in Th2 cells (25). The mechanisms of glucocorticoid action have been attributed to gene expression of anti-inflammatory genes such as annexin-1, IL-10, and inhibitors of the inflammatory transcription factor NF-{kappa}B (25). These are cellular systems that are known to be modified by zinc (13, 24, 26, 42). Thus it will be of interest to verify whether our observation that cortisol increases zinc influx in gill epithelial cells also occurs in other cell types, such as the lung.

In conclusion, the present study demonstrates that cortisol-induced expression of MTA, MTB, and ZnT1 in fish gill cells is due to cortisol stimulation of zinc influx and subsequent activation of the zinc MTF1 pathway. The observation that cortisol can mediate zinc influx may be of significance in other cells, such as the lung and immune system, where cross talk between the GR and zinc signaling may play an important role in modifying the effects of gene expression.


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 GRANTS
REFERENCES
 
This work was supported by research (NER/A/S/2000/00550) and studentship (NER/S/J/2000/05957) grants from the Natural Environment Research Council to C. Hogstrand and by Research Grant S18960 from the Biotechnology and Biological Sciences Research Council to N. R. Bury.


   ACKNOWLEDGMENTS
 
Present addresses: M. J. Chung. The Nutraceutical BioBrain Korea 21 Project Group, Kangwon University, Chuncheon 200-701 Korea. P. A. Walker, Cells to Tissues, University of Manchester, Smith Building, Oxford Road, Manchester M13 9PT, UK; A. Sturm, Institute of Aquaculture, University of Stirling, Stirling FK9 4LA, UK.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. Hogstrand, King's College London, Nutritional Sciences Division, Franklin-Wilkins Bldg., 150 Stamford St., London, SE1 9NH, UK (e-mail: christer.hogstrand{at}kcl.ac.uk)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Andrews GK. Regulation of metallothionein gene expression by oxidative stress and metal ions. Biochem Pharmacol 59: 95–104, 2000.[CrossRef][Web of Science][Medline]
  2. Andrews GK. Cellular zinc sensors: MTF1 regulation of gene expression. Biometals 14: 223–237, 2001.[CrossRef][Web of Science][Medline]
  3. Auf Der Maur A, Belser T, Elgar G, Georgiev O, Schaffner W. Characterization of the transcription factor MTF1 from the Japanese pufferfish (Fugu rubripes) reveals evolutionary conservation of heavy metal stress response. Biol Chem 380: 175–185, 1999.[CrossRef][Web of Science][Medline]
  4. Balesaria S, Hogstrand C. Identification, cloning and characterization of a plasma membrane zinc efflux transporter, TrZnT-1, from fugu pufferfish (Takifugu rubripes). Biochem J 394: 485–493, 2006.[CrossRef][Web of Science][Medline]
  5. Beltramini M, Zambenedetti P, Wittkowskim W, Zatta P. Effects of steroid hormones on the Zn, Cu and MTI/II levels in the mouse brain. Brain Res 1013: 134–141, 2004.[CrossRef][Web of Science][Medline]
  6. Bonewitz RF, Foulkes EC, O'Flaherty EJ, Hertzberg VS. Kinetics of zinc absorption by the rat jejunum: effects of adrenalectomy and dexamethasone. Am J Physiol Gastrointest Liver Physiol 244: G314–G320, 1983.[Abstract/Free Full Text]
  7. Bonham K, Zafarullah M, Gedamu L. The rainbow trout metallothioneins: molecular cloning and characterization of two distinct cDNA sequences. DNA 6: 519–528, 1987.[Web of Science][Medline]
  8. Brugnera E, Georgiev O, Radtke F, Heiuchel R, Baker E, Sutherland GR, Schaffner W. Cloning, chromosomal mapping and characterization of the human metal-regulatory transcription factor MTF1. Nucleic Acids Res 22: 3167–3173, 1994.[Abstract/Free Full Text]
  9. Bury NR. The changes to apical silver membrane uptake, and basolateral membrane silver export in the gills of rainbow trout (Oncorhynchus mykiss) on exposure to sublethal silver concentrations. Aquat Toxicol (Amst) 72: 135–145, 2005.[CrossRef]
  10. Bury NR, Sturm A, Le Rouzic P, Lethimonier C, Ducouret B, Guiguen Y, Robinson-Rechavi M, Laudet V, Rafestin-Oblin ME, Prunet P. Evidence for two distinct functional glucocorticoid receptors in teleost fish. J Mol Endocrinol 31: 141–156, 2003.[Abstract]
  11. Carpene E, Camatti A, Isani G, Cattani O, Cortesi P. Cd-metallothionein in liver and kidney of goldfish (Carassius auratus): effects of temperature and salinity. Ital J Biochem 41: 273–282, 1992.[Web of Science][Medline]
  12. Chung MJ, Walker PA, Brown RW, Hogstrand C. ZINC-mediated gene expression offers protection against H2O2-induced cytotoxicity. Toxicol Appl Pharmacol 205: 225–236, 2005.[CrossRef][Web of Science][Medline]
  13. Cousins RJ, Blanchard RK, Popp MP, Liu L, Cao J, Moore JB, Green CL. A global view of the selectivity of zinc deprivation and excess on genes expressed in human THP-1 mononuclear cells. Proc Natl Acad Sci USA 100: 6952–6957, 2003.[Abstract/Free Full Text]
  14. Cox RP, Ruckenstein A. Studies on the mechanism of hormonal stimulation of zinc uptake in human cell cultures: hormone-cell interactions and characteristics of zinc accumulation. J Cell Physiol 77: 71–81, 1971.[CrossRef][Web of Science][Medline]
  15. Dalton TP, Solis WA, Nebert DW, Carvan MJ. Characterization of the MTF1 transcription factor from zebrafish and trout cells. Comp Biochem Physiol B 126: 325–335, 2000.[CrossRef][Medline]
  16. Ducouret B, Tujague M, Ashraf J, Mouchel N, Servel N, Valotaire Y, Thompson EB. Cloning of a teleost fish glucocorticoid receptor shows that it contains a deoxyribonucleic acid-binding domain different from that of mammals. Endocrinology 136: 3774–3783, 1995.[Abstract]
  17. Feeney GP, Zheng DL, Kille P, Hogstrand C. The phylogeny of teleost ZIP and ZnT zinc transporters and their tissue specific expression and response to zinc in zebrafish. Biochim Biophys Acta 1732: 88–95, 2005.[Medline]
  18. Giedroc DP, Chen X, Apuy JL. Metal response element (MRE)-binding transcription factor-1 (MTF1): structure, function, and regulation. Antioxid Redox Signal 3: 577–596, 2001.[CrossRef][Web of Science][Medline]
  19. Grosell. M, Hansen HJ, Rosenkilde P. Cu uptake, metabolism and elimination in fed and starved European eels (Anguilla anguilla) during adaptation to water-borne Cu exposure. Comp Biochem Physiol C 120: 295–305, 1998.[Web of Science][Medline]
  20. Hager LJ, Palmiter RD. Transcriptional regulation of mouse liver metallothionein-I gene by glucocorticoids. Nature 291: 340–342, 1981.[CrossRef][Medline]
  21. He P, Xu M, Ren H. Cloning and functional characterization of 5'-upstream region of metallothionein-I gene from crucian carp (Carassius cuvieri). Int J Biochem Cell Biol 39: 832–841, 2007.[CrossRef][Web of Science][Medline]
  22. Herbomel P, Bourachot B, Yaniv M. Two distinct enhancers with different cell specificities coexist in the regulatory region of polyoma. Cell 39: 653–662, 1984.[CrossRef][Web of Science][Medline]
  23. Hogstrand C, Reid S, Wood CM. Ca2+ versus Zn2+ transport in the gills of freshwater rainbow trout and the cost of adaptation to waterborne Zn2+. J Exp Biol 198: 337–348. 1995.[Web of Science][Medline]
  24. Ibs KH, Rink L. Zinc-altered immune function. J Nutr 133: 1452S–1456S, 2003.[Abstract/Free Full Text]
  25. Ito K, Chung KF, Adcock IM. Update on glucocorticoid action and resistance. J Allergy Clin Immunol 117: 522–543, 2006.[CrossRef][Web of Science][Medline]
  26. Kabu K, Yamasaki S, Kamimura D, Ito Y, Hasegawa A, Sato E, Kitamura H, Nishida K, Hirano T. Zinc is required for Fc epsilon RI-mediated mast cell activation. J Immunol 177: 296–305, 2006.
  27. Kang YJ. Metallothionein redox cycle and function. Exp Biol Med (Maywood) 231: 1459–1467, 2006.[Abstract/Free Full Text]
  28. Karin M, Herschman HR, Weinstein D. Primary induction of metallothionein by dexamethasone in culture rat hepatocytes. Biochem Biophys Res Commun 92: 1052–1069, 1980.[CrossRef][Web of Science][Medline]
  29. Kelly EJ, Sandgren EP, Brinster RL, Palmiter RD. A pair of adjacent glucocorticoid response elements regulate expression of two mouse metallothionein genes. Proc Natl Acad Sci USA 94: 10045–10050, 1997.[Abstract/Free Full Text]
  30. Kelly SP, Fletcher M, Part P, Wood CM. Procedures for the preparation and culture of "reconstructed" rainbow trout branchial epithelia. Methods Cell Sci 22: 153–163, 2000.[CrossRef][Medline]
  31. Kille P, Kay J, Sweeney GE. Analysis of regulatory elements flanking metallothionein genes in Cd-tolerant fish (pike and stone loach). Biochim Biophys Acta 1216: 55–64, 1993.[Medline]
  32. Lang C, Murgia C, Leong M, Tan LW, Perozzi G, Knight D, Ruffin R, Zalewski P. Anti-inflammatory effects of zinc and alterations in zinc transporter mRNA in mouse models of allergic inflammation. Am J Physiol Lung Cell Mol Physiol 292: L577–L584, 2007.[Abstract/Free Full Text]
  33. Mayer GD, Leach A, Kling P, Olsson PE, Hogstrand C. Activation of the rainbow trout metallothionein-A promoter by silver and zinc. Comp Biochem Physiol B 134: 181–188, 2003.[CrossRef][Medline]
  34. Muto N, Ren HW, Hwang GS, Tominaga S, Itoh N, Tanakam K. Induction of two major isoforms of metallothionein in crucian carp (Carassius cuvieri) by air-pumping stress, dexamethasone, and metals. Comp Biochem Physiol C 122: 75–82, 1999.[Web of Science][Medline]
  35. Oliver PD, Tate DJ, Newsome DA. Metallothionein in human retinal pigment epithelial cells: expression, induction and zinc uptake. Curr Eye Res 11: 183–188, 1992.[CrossRef][Web of Science][Medline]
  36. Olsson PE, Hyllner SJ, Zafarullah M, Andersson T, Gedamu L. Differences in metallothionein expression in primary cultures of rainbow trout hepatocytes and the RTH-149 cell line. Biochim Biophys Acta 1049: 78–82, 1990.[Medline]
  37. Olsson PE, Kling P, Erkell LJ, Kille P. Structural and functional analysis of the rainbow trout (Oncorhyncus mykiss) metallothionein-A gene. Eur J Biochem 230: 34924–34928, 1995.
  38. Palmiter RD. The elusive function of metallothioneins. Proc Natl Acad Sci USA 95: 8428–8430, 1998.[Abstract/Free Full Text]
  39. Pattison SE, Cousins RJ. Kinetics of zinc uptake and exchange by primary cultures of rat hepatocytes. Am J Physiol Endocrinol Metab 250: E677–E685, 1986.[Abstract/Free Full Text]
  40. Quaife CF, Findley SD, Erickson JC, Froelick GJ, Kelly EJ, Zambrowicz BP, Palmiter R. D, Induction of a new metallothionein isoform (MT-IV) occurs during differentiation of stratified squamous epithelia. Biochemistry 33: 7250–7259, 1994.[CrossRef][Web of Science][Medline]
  41. Ren H, Xu M, He P, Muto N, Itoh N, Tanaka K, Xing J, Chu M. Cloning of crucian carp (Carassius cuvieri) metallothionein-II gene and characterization of its gene promoter region. Biochem Biophys Res Commun 342: 1297–1304, 2006.[CrossRef][Web of Science][Medline]
  42. Rink L, Haase H. Zinc homeostasis and immunity. Trends Immunol 28: 1–4, 2007.[CrossRef][Web of Science][Medline]
  43. Roesijadi G. Metallothionein induction as a measure of response to metal exposure in aquatic animals. Environ Health Perspect 102: 91–95, 1994.
  44. Sabourin TD, Gant DB, Eber LJ. The influence of metal and nonmetal stressors on hepatic metal-binding protein production in Buffalo sculpin, Enophrys bison. In: Marine Pollution and Physiology: Recent Advances, edited by Vernberg FJ, Thurberg FP, Calabrese A, Vernberg W. Columbia, SC: South Carolina Press, 1985, p. 247–266.
  45. Samson SL, Gedamu L. Metal-responsive elements of the rainbow trout metallothionein-B gene function for basal and metal-induced activity. J Biol Chem 270: 6864–6871, 1995.[Abstract/Free Full Text]
  46. Samson SL, Paramchuk WJ, Gedamu L. The rainbow trout metallothionein-B gene promoter: contributions of distal promoter elements to metal and oxidant regulation. Biochim Biophys Acta 1517: 202–211, 2001.[Medline]
  47. Saydam N, Georgiev O, Nakano MY, Greber UF, Schaffner W. Nucleo-cytoplasmic trafficking of metal-regulatory transcription factor 1 is regulated by diverse stress signals. J Biol Chem 276: 25487–25495, 2001.[Abstract/Free Full Text]
  48. Saydam N, Steiner F, Georgiev O, Schaffner W. Heat and heavy metal stress synergize to mediate transcriptional hyperactivation by metal-responsive transcription factor MTF1. J Biol Chem 278: 31879–31883, 2003.[Abstract/Free Full Text]
  49. Schulte PM, Glemet HC, Fiebeg AA, Power DA. Adaptive variation in the lactate dehydrogenase-B expression: Role of a stress-responsive regulatory element. Proc Natl Acad Sci USA 97: 6597–6602, 1997.[CrossRef]
  50. Scudiero R, Carginale V, Capasso C, Riggio M, Filosa S, Parisi E. Structural and functional analysis of metal regulatory elements in the promoter region of genes encoding metallothionein isoforms in the Antarctic fish Chionodraco hamatus (icefish). Gene 274: 199–208, 2001.[CrossRef][Web of Science][Medline]
  51. Sogawa CA, Asanuma M, Sogawa N, Miyazaki I, Nakanishi T, Furuta H, Ogawa N. Localization, regulation, and function of metallothionein-III/growth inhibitory factor in the brain. Acta Med Okayama 55: 1–9, 2001.[Medline]
  52. Sturm A, Bury NR, Dengreville L, Fagart J, Flouriot G, Rafestin-Oblin ME, Prunet P. 11-deoxycorticosterone is a potent agonist of the rainbow trout (Oncorhynchus mykiss) mineralocorticoid receptor. Endocrinology 146: 47–55, 2005.[Abstract/Free Full Text]
  53. Vasak M. Advances in metallothionein structure and functions. J Trace Elem Med Biol 19: 13–17, 2005.[CrossRef][Web of Science][Medline]
  54. Walker PA, Bury NR, Hogstrand C. Influence of culture conditions on metal-induced responses in a cultured rainbow trout gill epithelium. Environ Sci Technol 15: 6505–6513, 2007.
  55. Wendelaar Bonga SE. The stress response in fish. Physiol Rev 77: 591–625, 1997.[Abstract/Free Full Text]
  56. Zhang B, Egli D, Georgiev O, Schaffner W. The Drosophila homolog of mammalian zinc finger factor MTF1 activates transcription in response to heavy metals. Mol Cell Biol 21: 4505–4514, 2001.[Abstract/Free Full Text]




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