Europ. J. Protistol, 36,437-442 (2000)December 29, 2000http://www.urbanfischer.de/journals/ejp

European Journal of

PROTISTOLOGY

Regulation of Metallothionein (MT) inTetrahymena: Induction of MT-mRNA andProtein by Cadmium Exposure

Gianfranco Santovito, Paola Irato and Ester Piccinni"

Department of Biology, University of Padua, ViaU. Bassi 58/B, 1-35131 Padova, Italy; Fax: 39-049-8276300;e-mail: piccinni@civ.bio.unipd.it

Summary

Cd-dependent regulation of MT expression in Tetrahymenapigmentosa and T. pyriformis was investigated by measur­ing MT-mRNA and MT accumulation in response tochronic exposure to subtoxic doses of the metal. MT syn­thesis occurs very soon and MT content increases progres­sivelywith Cd accumulation in both species. The inductionof MT-mRNA in response to Cd is very rapid, with no ob­servable lag period. At the beginning of exposure, verysmall amounts of intracellular Cd are sufficient to induce astrong response. Maximum levels of MT-mRNA induc­tion, more than 40-fold, occurred within 30 min of metaltreatment in T. pigmentosa. This maximum induction wasthen followed by down-regulation to 20-fold above theoriginal basal level over the next 30 min. In T. pyriformis,the MT-mRNA maximum levelwas reached after 60 min,followed by a slow decrease to about 25-fold the basal levelwithin 24 h. This transient fluctuation of mRNA levels issimilar to those in other organisms. The data emphasisethe importance of an efficient detoxification pathway bycomplex Cd regulation of MT transcription, which enablesTetrahymena to survive in the continued elevated presenceof toxic ions in the environment.

Key words: Cadmium; Gene expression; Metallothionein,Tetrahymena.

Introduction

Metallothioneins (MTs), a family of characteristical­ly low molecular weight, and heavy metal-binding pro­teins, are found in most organisms. A considerableamount of work has been done on the induction ofthese proteins by many agents, especially heavy metals,

"corresponding author

© 2000 by Urban & Fischer Verlag

in various animals and tissues [7]. Much of our presentknowledge of MT gene structure and regulatory mech­anisms are from mammals, such as mouse and humanor the yeast Saccharomyces. So far only one Cd-MT andits gene sequence have been described in unicellularprotist organisms.

When Tetrahymena pigmentosa or T pyriformis areexposed to sub toxic doses of cadmium, a Cd-metal­lothionein accumulates in the cells. This polypeptide iscysteine-rich (30%), but has only limited similaritywith other MTs. It is unusually long, having 107 aminoacids, and besides the characteristic Cys-X-Cys andCys-Cys motifs, it exhibits four groups of Cys in anatypical context, Cys-Cys-Cys [15]. The genome se­quence encoding this Cd-MT has recently been de­scribed [13]. Southern blot analyses suggest thatTetrahymena has only one functional copy of the Cd­MT gene and no pseudogenes. The gene encodes a tran­script of 487 bases, with an intronless coding region of324 nucleotides. As expected, the translational termina­tion codon is TGA, the only codon used as a stop inTetrahymena. Comparisons between Cd-MT 3'-VTRand 5' -VTR gene regions with those of known pluricel­lular eukaryotes show a slight degree of similarity. Nei­ther GC or CCAAT boxes nor typical MRE sequencesare detected in 525 bp upstream from the transcription­al initiation site. However, a putative TATA box is pre­sent at 44 bp upstream from the initiation of transcrip­tion, and a putative CAAT box, TTGGAT, the reversecomplement of the consensus sequence core of ciliateCCAAT box [1] is present at nt-104 to -99.

In order to study Cd-dependent regulation ofTetrahymena MT expression, we investigated MT­mRNA induction and MT accumulation in response tochronic exposure to subtoxic doses of Cd in both Tpigmentosa and T. pyriformis.

0932-4739/00/36/04-437 $ 15.00/0

438 G.Santovito, P. Irataand E. Piccinni

The data presented here support the hypothesis of anefficient Cd ion transport and detoxification pathway,which enables Tetrahymena to survive in the continuedelevated presence of toxic ions in the environment.

Material and Methods

Tetrahymena thermophila 17S rRNA oligonucleotide probelabelled with [y-32P]dATP [5] was used to normalise RNAloading [18].Hybridisation signals were detected and quanti­fied by exposure to a Packard Instant Imager A2024. All blotswere also exposed to X-ray films. For dot blot analyses,nylon membranes were loaded in triplicate with 5-Jlg por­tions of total denatured RNA. The membranes were then hy­bridised with the same probe used for Northern blot analy­ses.

Cell growth and Cd treatment. T. pyriformis (strain GL)and T. pigmentosa (mating type 8) were grown axenically ac­cording to standard methods [16], as previously described[15]. Treated cells were grown in the same medium supple­mented to a final non-toxic Cd concentration of 44.5 JlM[12].For the dose-response experiment, cells were also exposed to26.7 and 62.3JlMCd.

Quantification of MT and Cd in cell-free extracts. Cellswere harvested by centrifugation at 2,000xg for 15 min at theindicated times, then homogenised as previously described[14]. The homogenates were centrifuged at 48,000xg for 50min at 4°C, and the resulting supernatants were used for MTand Cd quantification. MT concentration was determined bythe silver saturation method [17]. Cd analysis was performedby atomic absorption spectrophotometer (Perkin-Elmermod. 4000). Data refer to total protein concentration, assayedby the Folin phenol reagent method [9].All results are report­ed as means ± standard deviation. Linear regression was car­ried out to estimate the correlation between MT and Cd con­centrations.

RNA isolation and MT-mRNA analyses. Total RNAwas isolated using Trizol reagent (LifeTechnologies, Gaithes­burg, MD, USA). RNA integrity was judged by visualisationof rRNA in ethidium bromide-stained gels. For Northernblot analyses, 2-Jlgportions of total RNA were size-fraction­ated on 1.2% formaldehyde agarose gelsand blotted on nylonmembranes. The membranes were hybridised with theeDNA probe corresponding to the coding region of Tetra­hymena Cd-MT gene [13] and labelled with [a-32P]dATP [5].For radioactive labelling, the membranes were hybridisedusing the solution suggested by Church and Gilbert [3] forabout 15 h at 65°C, and washed twice in 0.1 M sodium phos­phate buffer (pH 7.0) and 1% SDS for 10 min at 60°C.

Results

Cd and MTaccumulation

Cd accumulation measured in the cell free extracts ofT pigmentosa was time-dependent. A high rate of as­sumption was evident after 4 h of exposure to a non­toxic dose of 44.5 pM metal. At 48 h, the amount in­creased by three times, remaining in steady state overthe next 24 h (72 h). Cd increased MT concentrationsand the time course of MT protein production indicat­ed that the protein was already present 30 min after thebeginning of Cd treatment; subsequently, its contentrapidly increased up to 48 h. In this organism, in whichthe time-course study was prolonged to 72 h, the 48 hvalue was maintained until 72 h (Fig. lA). The correla­tion index between Cd and Cd-MT contents was 0.991(p < 0.001) (Fig. lB).

As expected also in T pyriformis cells, Cd accumula­tion was time-dependent, and a very high rate of as­sumption was evident after 4 h exposure; at 48 h theamount increased by three times. The time course ofMT concentration indicated that the protein was pre­sent 1 h after the beginning of treatment, and then in­creased, mostly after 4 h (Fig. 2A). The correlationindex between Cd and Cd-MT was 0.993 (p < 0.001)(Fig.2B).

Fig. 1. Kinetics of Cd and MT accumulation in T. pig­mentosa. A, cells cultured in 44.5 ]JMCd and harvestedat 30 min, 1,2,4,24,48 and 72 h. Control cells harvestedat 1,4,24 and 48 h. -0- MT in control cells;-e- MT intreated cells;-0- Cd in control cells;-.- Cd in treatedcells. B, correlation between Cd and MT concentrationsin cell-free extracts of treated cells.

B

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72h4h 24h 48h

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Regulation of Cd-Metallothionein in Tetrahymena 439

A B

35

30

.~ 25e2- 20oCl

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5

15' 30' 1h 2h

5 .s 35 . /~ 30 r =0.99T 5. 25 p c 0.001 /•

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3 K• ~ 5 I '·.. / "" '0 Cl 0::1Cl 0 1 2 3 4E• 2 :0 lJgCd/mg of protein

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Fig. 2. Kinetics of Cd and MT accumulation in T. pyri-formis. A, control and treated cells harvested at 15, 30- - min, 1,2,4,24 and 48 h. -0- MT in control cells; -e-

==9= 0 MT in treated cells;-0- Cd in control cells;-.- Cd in4h 24h 48h treated cells. B, correlation between Cd and MT con-lime centrations in cell-free extracts of treated cells.

60' 48h 15' 48h 72h

MT induction and expression

In order to show the induction of MT-mRNA byCd, Northern blot analyses were performed using totalRNA from both T pigmentosa and T pyriformis con­trols and 44.5 pM Cd treated cells. Fig. 3A shows thatMT-mRNA was induced following Cd treatment. Inorder to determine the pattern of this increase overtime, an early time-course study was carried out, usingthe total RNA from both species.

Fig. 3A and 4A show Northern blot analyses of Cd­treated T pigmentosa at different time-points, in whicha 175 rRNA probe was used to normalise RNA loading(Fig. 3B, 4B). Quantifications are shown in Fig. SA,which also indicates that some hybridisation took placevery quickly and increased with minutes after exposureto metal. The maximum level of MT-mRNA induction,more than 40-fold, occurred within 30 min of metaltreatment. This maximum induction was then followedby down-regulation to 20-fold above the original basallevel over the next 30 min. A further 35-fold inductionthen occurred within 2 h. This steady level was main­tained for 24 h, followed by another down-regulationand a further increase over the next two days (at 48 and72 h respectively). The concentration response of MT­mRNA induction observed at 30 min indicated thatmaximal production occurs at doses of 5-7 pg Cd/ml(44.5-62.3 pM) (Fig. 6).

2h 4h 24h 48h1h30'

A

Cd-treatedControls

A

8

B

Fig.3. Northern blot analysis of T. pigmentosa. A, samples of2}lg of total RNA hybridised with Cd-MT eDNA probe. B,re-hybridisation with T. thermophila 175 rRNA oligonu­cleotide probe.Control and treated cells harvested after indicated times.

Fig. 4. Northern blot analyses of T. pigmentosa. A, samplesof 2 }lg total RNA were hybridised with Cd-MT eDNAprobe. B, re-hybridisation with T. thermophila 175 rRNAoligonucleotide probe.Cells harvested after indicated times.

440 G. Santavita, P. Irataand E. Piccinni

The same time-course analyses performed on T.pyriformis showed that MT-mRNA accumulationpeaked at 60 min, followed by a slow decrease to about25-fold within 24 h. Induction then increased to valuenear the maximum at 48 h (Fig. 5B).

A 6

Control

Cd-treated5 min

T.'

6

Cd-treatedControl

Fig. 7. Dot blot analysis of control and 5 min Cd-treatedT. pyriformis cells. 5 JIg of total RNA loaded in triplicate andhybridised, as described in Material and Methods.

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48h

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Fig. 5. Time course of Cd-MT mRNA induction. Values ex­pressed as arbitrary units (a.u.), normalised against those forT. thermophila 175 rRNA. A, T. pigmentosa. B, T. pyriformis.

oC 30' 1h 2h 4h 24h

Time48h

Fig.8. Northern blot analysis of total RNA from control andCd-treated cells of T. pigmentosa after 48 h of growth. 15 JIgof total RNA loaded in each line.

Fig. 6. Dose-dependent MT-mRNA induction by Cd in T.pigmentosa. Quantification values, expressed in arbitraryunits (a.u.), determined after 30 min exposure. Hybridisationand normalisation of total RNA made as described in Materi­al and Methods.

5

4

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Dot blot analyses were also made for both species afew minutes after Cd exposure. Fig. 7, referring to T.pyriformis, shows that some hybridisation also oc­curred 5 min after metal treatment. A weak signal couldalso be highlighted in control cells of both species,using 15 pg total RNA, corresponding to the basal levelof the MT-mRNA. Fig. 8 shows this hybridisation sig­nal in T. pigmentosa.

Discussion

This study reports the only data presented until nowon MT gene expression in protists after Cd exposure. Itis evident that MT contents increase progressively withCd accumulation in both T. pigmentosa and T. pyri­formis cultured in vitro, the correlation index betweenmetal and protein indicating that all Cd present in cellfree-extracts is linked to MTs. MT synthesis occursvery soon in both protists, within 1 h after the begin­ning of Cd exposure (Fig. lA, 2A). Then a clearly dis-

tinguishable increase occurs, and protein levels are in­duced 3D-fold at 48 h. The 3-day time-course studyperformed on T pigmentosa indicates that MT contentsteady state corresponds to the beginning of stationaryphases of culture at 48 h [12].

A concentration-dependent increase in MT-mRNAwas observed up to 5 pg Cd/ml (44.5 pM). In both Tpigmentosa and T pyriformis, the increase in MT­mRNA in response to Cd is very rapid (Fig. 5), with noobservable lag period within the time points tested.This is confirmed by dot blot analyses which showsome hybridisation even 5 min (Fig. 7). We also demon­strated that, in the chronic elevated presence of non­toxic doses of a toxic metal like Cd, maximum induc­tion is reached within 1 h. This pathway is faster than inany mammalian system, including culture cells, and issimilar to that of Neurospora [10] and Saccharomyces[11]. This maximum induction is followed by repres­sion, especially evident in T pigmentosa, in which twostrong but transient down-regulations occur during thecourse of 72 h. However, the MT-mRNA level neverdecays to basal level, but it is maintained at relativelyhigh levels with increasing mRNA expression after 1and 48 h.

In T pyriformis, the MT-mRNA level is not signifi­cantly down-regulated after initial maximum inductionand is maintained between 25- and 40-fold the basallevel from 2 to 48 h.

Previous studies in pluricellular organisms haveshown that administration of Cd transiently inducesMT-mRNA transcription. In mice liver and kidney,maximum MT-mRNA levels are observed 4-6 h aftermetal administration and then fall within 9-12 h [2, 4].In turbot, the response depends on Cd dose and tissuesinvolved, maximum MT-mRNA levels being attainedbetween 1 and 2 days in kidney and about 4 days inliver. The levels then fall within 21 days but, at a moder­ately hepatotoxic dose of Cd, hepatic MT-mRNA re­mains at high levels for at least 21 days [6]. In the abovestudies, however, MT induction was measured in re­sponse to a single injection of Cd. This initial transientactivation has also been found in mammal cell culturesand in unicellular organisms such as Saccharomyces [11]and Neurospora [10] exposed to metals over all the timeperiods examined. In HeLa cells, the increase in MT­mRNA in response to Zn is very rapid with no observ­able lag period: the maximum level occurs at 8 h butthen declines to about 30% of the peak 24 h after induc­tion [8]. In Hepa lA cells treated with Zn, a decline inMT-mRNA levels are frequently observed after an ini­tial peak at 6-8 h [19]. In Neurospora, Cu treatmentstrongly induces Cu-MT-mRNA levels within 1 h fol­lowed by repression to basal levels within 8 h, whereasthe Cu-MT protein peaks within 3 h and remains at thesame elevated levels over 17 h [10].

Regulation of Cd-Metallothionein in Tetrahymena 441

In Saccharomyces, the chronic and elevated presenceof Cu ions induces a rapid (within 30 min) but transientAce1p-mediated transcriptional activation of CUP1­mRNA expression, followed by a precipitous reduc­tion within 60 min. The authors demonstrate that thismechanism reflects fluctuations in the availability of in­tracellular Cu ions, the transport of which is regulatedby different sensors to maintain appropriate intracellu­lar Cu levels [11].

Our results show that continued Cd uptake allowsTetrahymena to grow normally in the presence of44.5 pM Cd [12]. Consistent with this high tolerance isa linear increase in MT accumulation within two days.The transient fluctuations of mRNA levels remain to beexplained. Unfortunately, cis acting and trans acting el­ements for metal regulation still remain to be identifiedin Tetrahymena. Characteristic sequences present inthe MT promoters of other organisms have not beenfound in this ciliate. Only short stretches partiallymatching the AP1 and ACEI binding sites have beenfound in proximal DNA upstream from the transcrip­tion site [13]. However, our present results show that,at the beginning of exposure, very low amounts of in­tracellular Cd are sufficient to induce a strong responseresulting in very high levels of MT-mRNA. Assumingthat regulation of MT genes is mediated by a metal reg­ulatory proteinlfactor (MTF), the increased synthesisof MT in Tetrahymena sequesters Cd, rendering theMTF less able to bind to the MRE providing mecha­nism for auto-regulating its own transcription. Thismechanism for Cd detoxification by MT requires addi­tional Cd uptake.

The data presented here emphasise the importance ofcomplex Cd regulation of MT-gene transcription,which creates an efficient detoxification pathway al­lowing Tetrahymena to survive in the continued elevat­ed presence of toxic ions in the environment.

Acknowledgements: This study was partly supported bya grant from the Italian Ministero Universita e Ricerca Scien­tifica Tecnologica. The authors are grateful to Professor Vin­cenzo Albergoni (Department of Biology, University ofPadova) for the constant interest and support.

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