In vitro effects of sodium selenite on nuclear 3,5,3'-triiodothyronine (T3) receptor gene expression...

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In Vitro Effects of Sodium Selenite on Nuclear 3,5,3'-Triiodothyronine

(T3) Receptor Gene Expression in Rat Pituitary GH4C1 Cells

JULIUS BRTKO,1, * PETER FILIP(:iK, 1 SONA HUDECOV,~,, 2 VLADIMiR ~TRB,~K, 1 AND ANASTAZIA BRTKOV,~ 3

'Institute of Experimental Endocrinology, Slovak Academy of Sciences, Vl~rska 3, 833 06 Bratislava, 2 Department

of Molecular Biology, Faculty of Natural Sciences, Comenius University, Bratislava; and 3Research Institute of Nutrition,

BratBlava, Slovakia

Received August 19, 1993; Accepted June 1, 1994

ABSTRACT

The present study was undertaken in order to investigate the effects of sodium selenite on:

1. The growth of rat pituitary GH4C1 cells; 2. The nuclear T3 receptor gene expression; 3. The cytoplasmic protein phosphorylation; and 4. The prolactin secretion in rat pituitary GH4C1 cell line.

Sodium selenite (up to 2.5 W~r has no inhibitory effect on GH4C1 cell proliferation as well as the prolactin secretion. On the other hand, 0.5 ~M sodium selenite significantly decreases the rate of mRNA synthesis and/or degradation of both, the ~1 form of the T3 receptor (TR0fl) and the or isoform of the T3 receptor. At I WVI of sodium selenite, significant changes in the electrophoretic profile of low molecular mass cytoplasmic proteins were found, moreover, sodium selenite (1 W~I) also considerably affects phosphorylation of a higher molecular mass proteins. The results based on the in vitro experiments suggest that sodium selenite may affect specific processes at the pretranslational level as well as it may also take part in processes of posttranslational modification of protein(s), the cell vitality and the cell growth remaining unchanged.

*Author to whom all correspondence and reprint requests should be addressed.

Biological Trace Element Research 173 Vol. 48, 1995

174 Brtko et al.

Index Entries: Thyroid hormone receptor; selenite; GH4C1 pituitary tumor cells; gene expression; protein phosporylation.

INTRODUCTION

Selenium is a trace element that plays an important role in the human body. Its presence is necessary for many functions in the metabolism of higher organisms. In the first half of this century selenium was considered an undesirable element for humans mainly because of its tox- icity. A significant change of mind has taken place on the importance of selenium for the normal function of higher vertebrates because of fol- lowing findings:

1. Selenium was able to prevent liver necrosis in animals con- suming a vitamin E deficient diet (1).

2. Selenium is a part of enzyme glutathion peroxidase (E.C. 1.11.1.9.) that catalyzes the conversion of peroxides and hydroperoxides into water and corresponding alcohols, respectively (2,3).

3. Taken with the above data, selenium together with vitamin E play an important role as components in the antioxidant system of cells that eliminate or decrease toxic effects of free radicals.

4. An increased intake of selenium has been suggested to diminish the incidence of cancer diseases in humans or to prolong their incubation period by 90% when compared to control values (4).

The proposed mechanism for a selenium anticancerogenic effect include:

1. A decrease in the oxidant effect of cancerogens; 2. Protection from cell damage by oxygen radicals; 3. Modification of proteins; also it is suggested that a direct

binding of selenium to DNA may occur (5); 4. An increased activation of the immune system; several

recent data showed that a deficit of selenium in the organ- ism impairs the immune system (6).

A remarkable breakthrough in the field of endocrinology came with recent published data that brought strong evidence that the activity of iodothyronine 5'-deiodinase is dependent on selenium (7), moreover, with recent identification of 5'-deiodinase, type I as a selenoenzyme with a subunit molecular mass of 27.5 kDa (8-10). Last but not least, the fol- lowing important property of selenium is that in the form of selenite (Se W) it takes part in the oxidation of sulfhydryl groups of proteins (11). Tashima et al. (12) recently showed a significant effect of selenite on the binding characteristics of glucocorticoid receptors since the molecule of

Biological Trace Element Research VoL 48, 1995

Sodium Selenite and Rat Pituitary 175

the receptor contains sulfhydryl groups. Our recent findings showed that the thyroid hormone receptor is extremely sensitive to selenite, as well (13). On the basis of the data dealing with thyroid hormone receptors it has been shown that they are like glucocorticoid receptors, members of a group of nuclear proteins so-called "ligand responsive transcription factors" (14). In general, thyroid hormone receptors are classified into cx and ]3 subtypes based on mapping to human chromosomes and on sequence homology. Gene localization of the thyroid hormone receptor ~1 (TRo~I) corresponds to human chromosome 17 and the [3-forms of the thyroid hormone receptor (TR]~I, TR[~2) to human chromosome 3 (15). Further investigations led to discovery of the carboxy-terminal TRR2 variant of the T3 receptor that binds to DNA but it fails to bind thyroid hormone (16-18).

Here we report in vitro effects of sodium selenite (at the concentrations that have no effects on cell vitality, cell proliferation, or prolactin secretion) on gene expression of the nuclear TR0tl receptor and the TRy2 variant of the T3 receptor as well as on cytoplasmic protein phosphorylation in prolactin producing GH4C1 rat pituitary cell line.

MATERIALS AND METHODS

(~_[32p] dCTP (3000 Ci/mmole), ~_[32p] ATP (>5000 Ci/mmole), and Hybond N + membranes were purchased from Amersham (Aylesbury, UK). Sodium selenite, Dulbecco's modified Eagle's medium (high glu- cose concentration formulation) (DMEM), gentamicin, and most other chemicals were purchased from Sigma (St. Louis, MO). Pancreatic trypsin was purchased from Serva Feinbiochemicals (Heidelberg, Germany), fetal calf serum was obtained from Veterinary School Brno (Brno, Czech Republic). Chemicals for gel electrophoresis, Dowex 1-X 8 were from Bio- Rad Company (Richmond, CA), Polyfix-1000 from Desaga (Heidelberg, Germany) and plasticwares from Flow Laboratories Company (Rick- mansworth, UK).

Cell Cul ture

Rat pituitary GH4C1 cells were grown as monolayer culture in selenium free DMEM supplemented with 10% fetal calf serum, 50 ~tg/mL gentamicin at 37~ in a humidified incubator IR 1500 (Flow Laboratories, Rickmansworth, UK) in a 5% CO2 atmosphere. The cells were plated at a density of 10.0-25.0 x 103 cells/cm 2, in 24 well plates. The medium was exchanged 24 h after plating for medium supplemented with indicated concentrations of sodium selenite. After 48 h the prolactin was estimated in the removed medium and cells were harvested by trypsinization using 0.25% pancreatic trypsin in 125 mM EDTA. The cell growth rate was determined by cell counting.

Biological Trace Element Research Vol. 48, 1995

176 Brtko et al.

Phosphorylat ion and SDS-PAGE of Cytoplasmic Proteins

Cells were homogenized by sonication in ice-cold riTE buffer (250 mM sucrose, 10 mM Tris-HC1 pH 7.2, 1 mM EGTA; 1:6 w/v) with 0.1 mM PMSF according to Hoang and Bergeron (19). The homogenate was then ultracentrifuged at 104,000 g for 1 h. The final cytosol fraction was immediately used for assays or stored at -70~ in small aliquots. The reaction was carried out in a total vol of 200 ~tL containing 50 mM Hepes buffer, pH 7.0, 10 mM MgC12, and 10 mM NaF in the presence of 0.3 mg protein. The reaction was initiated by addition of 20 ~tL of 10 W~I ~/_[32p] ATP, and samples were incubated at 30~ for 6 min. The reaction was stopped by 100 ~tL of the solution containing 9% SDS, 30% glycerol, 8% ~-mercaptoethanol, 50 mM Tris-HC1, pH 6.8, and 0.05% Bromophenol blue. The mixture was heated for 3 min in a boiling water bath and after- ward a 20 ~tL aliquot was applied to 12.5% SDS polyacrylamide slab gels (20), except that the electrophoresis was performed on ultrathin slab gels (0.3 mm) fixed on glass plates by Polyfix-100. Immobilized gels were stained in 0.25% Coomassie blue, dried, and scanned with the enhanced laser densitometer, Ultroscan XL (LKB, Bromna, Sweden). Gels were then exposed on Kodak X~ film at room temperature and autoradiograms scanned with the laser densitometry.

Northern Blot Hybridization Analysis Total cytoplasmic RNAs were isolated from 1 mL of cell culture (106

cells) according to the method of Chirgwin et al. (21). Polyadenylated mRNAs were isolated on the oligo (dT) cellulose column (Pharmacia, Uppsala, Sweden). The polyadenylated mRNAs were denatured with 1M glyoxal and 50% dimethylsulfoxide at 50~ for 1 h. After denaturation was completed, 10 ~tg of each sample were electrophoresed in 1.5% agarose gels and transferred to Hybond N+ membrane (Amersham, Arlington Heights, IL). The Pvu II fragment from plasmid rbeA12-P500 that contained the neuronal c-erbA cDNA (nucleotide position 607-1113) was used as the random prime labeled probe (22). The hybridizations were performed in 50% formamide at 42~ overnight in a Techne auto- matic hybridizer (Cambridge, UK). The membranes were then washed twice in 2x SSC (lx SSC = 0.15M NaC1/0.015M Na citrate) and 0.1% SDS at room temperature, twice in lx SSC and 0.1% SDS at room temperature. The last wash was done in 0.1x SSC and 0.1% SDS at 50~ for 10 min. The membranes were then autoradiographed for 2 d.

Assay of Prolactin

Rat prolactin in the cultivation medium was determined by RIA using a kit supplied by the Rat Pituitary Hormone Program, NIADDK, NIH (Bethesda, MD).



Table 1 Effect of Different Sodium Selenite (SelV) Concentrations on Cell Proliferation

and Prolactin Secretion in GH4C1 Cells Treated for 24 and 48 ha

Without Se TM 1.0 WVI Se TM 2.5 WVI Se TM

Cell number PRL, Cell number PRL, Cell number PRL, Time, h per cm2 ng/mL per cm2 ng/mL per cm2 ng/mL

0 146000 17.70 146000 17.70 146000 17.70 • • • • • •

24 265330 26.11 253000 24.88 237330 23.77 • • • • • •

48 513660 38.53 491000 40.75 506500 38.07 • • • • • •

a_+ s t a n d a r d dev ia t ion .

Determination of Selenium

Selenium in fetal serum was determined by graphite-furnace atomic absorption spectrometry with deuterium background and a reduced pal- ladium modifier, according Jacobson and Lockitch (23). The measure- ments were carried out with an atomic absorption spectrometer Varian, type Spectr. AA-30 (Melbourne, Australia) fitted with an autosampler and a graphite tube atomizer GTA-96.

Estimation of Protein

The protein concentration was determined by the method of Lowry et al. (24) using human albumin as a standard.

RESULTS

We began our studies by examining the effect of different concentrations of sodium selenite on GH4C1 cell proliferation and prolactin secretion in order to find a concentration of sodium selenite that will not inhibit both the cell proliferation and the prolactin secretion. As shown in Table 1, sodium selenite (up to 2.5 WVI) has no inhibitory effect on GH4C1 cell proliferation as well as the prolactin secretion. At selenite concentration of 5 wV/ and above, a significant inhibition of prolactin secretion and cell proliferation was observed (data not shown).

Effect of Sodium Selenite on Expression of the Nuclear I"3 Receptor in GH4CI Cells

The significant changes in the T3 receptor formation induced by selenite were found in the T3 receptor o~1 messenger RNA levels after 48

Biological Trace Element Research VoL 48, 1995

178 Brtko et al.

TRu (c-erbAa)

(6.0 kb) a l

(2.6 kb) a2

1 2 3 4 5 6 7 8 9

Fig. 1. The effect of 0 ~, i (1-3), 0.5 ~M (4--6), and 1.0 (7-9) sodium selenite on the expression of the nuclear T3 receptor ~1 (TRod) and its variant (TRy2) in GH4C1 cells (Northern blots). Cells were grown in the presence of the above concentrations of sodium selenite for 48 h. The length of polyadenylated mRNAs was estimated according to positions of E. coli rRNAs (Serva, Germany) and 28S/18S rRNAs in the gel.

h. Sodium selenite at 0.5 WV/acts at the pretranslational level to decrease significantly the rate of synthesis and/or increase the degradation of the thyroid hormone receptor (TRot) mRNA. Similar effect of 0.5 ~M sodium selenite on TRR2 mRNA synthesis and/or degradation was also confirmed (Figs. 1 and 2).

Effect of Sodium Selenite on Electrophoretic Profile of Cytoplasmic Proteins and Phosphorylation in GH4C1 Cells

As shown in Fig. 3, 1 pJVl selenite causes significant changes in the numbers of low molecular mass cytoplasmic proteins in the rat pituitary GH4C1 cells. Significant changes in the cytoplasmic protein phosphorylation predominantly of the higher molecular mass cytoplasmic proteins are shown in Fig. 4.

In order to know the absolute value of selenium content in the cultivation medium, the selenium concentration in fetal calf serum used in the experiments with the GH4C1 cells was determined. We found that a commercially available FCS contained 0.288 + 0.004 ~ selenium, i.e., the cultivation medium (selenium-free DMEM supplemented with 10% FCS) contained the lowest possible amount of 0.0288 + 0.0004 ~ (2.272 + 0.036 ng/mL) selenium. This value represents the concentration of selenium in the cultivation medium before being supplemented by the indicated (0.5, 1.0, or 2.5 gM) concentrations of sodium selenite.



";120

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60

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Fig. 2. Quantitative evaluation of autoradiograms (shown in Fig. 1) from Northern blots of polyadenylated mRNAs by laser densitometry (A: TR(~I; B: TR(~2; *p < 0.05).

DISCUSSION

According to reported data, the selenium concentration (predominantly in the form of SE-II) in normal adult blood is in the range 60-355 ng/mL, in selenium-deficient patients it is around 10 ng /mL (25). In our experiments, three concentrations of selenium, in the form of selenite (SEW) were used as follows: 0.5 ~ (39.48 ng/mL), 1.0 W~I (78.96 ng/mL), or 2.5 ~ (197.40 ng/mL). Golczewski and Frenkel (26) reported that sodium selenite (up to 1.0 ~M) stimulated proliferation in HeLa and HT-29 cells in a defined (serum free) medium in a gradual dose-dependent manner. This stimulation may not be detected in serum- supplemented media (26). Our findings of growth inhibition in GH4C1 cells at 5 W~I sodium selenite and above agree with the concentrations been reported previously (26,27). According to Morrison and Medina (28), the selenite metabolism in confluent cultures of mouse mammary epithelial cell (and probably in other cells, as well) involves steps as follows: Selenite is taken up by the cell and reduced by GSH and glu- tathione reductase to form HSe-. Then HSe- is used by a cytosolic enzyme to modify phosphoryl serine in the suppressor tRNA. This modification yields in part the selenonucleic acid. The rest of the selenonucleic acid fraction is made up of seleno-tRNAs (29). The sele- nocysteine-suppressor tRNA then suppresses the appropriate UGA codon allowing synthesis of a selenoprotein. One of the selenoproteins (58 kDa) may act as a direct or indirect inhibitor of DNA synthesis (28). The findings reported here suggest that selenium in the form of selenite may also act on specific gene expression. Our results demonstrate more than 80% inhibition of the TR0cl and more than 50% inhibition of the TR0c2 mRNAs accumulation by 0.5 wVI sodium selenite, i.e., at the con-


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kDa ~ 20.1 30,0 43,0 67.0 IMk0

A C :2":~T:::'.':~2":~7":~7"::"::?':7"Z"ZTZ?72"~Z'ZTZTZ?ZIZCZ'~

~..~-.-.~..~..~..-~.......~..~.~..~....~.~.......~...~..~.~.....~..~ ~ ............ 14A a~l ~:o" :~"" ~o'" "~o

Fig. 3. The effect of L0 I ~ sodium selenite on SDS-12.5% PAGE profile of cytoplasmic proteins. GH4C1 cells were grown in the presence of 1.0 ~ / i sodium selenite for 48 h. Cells were then soni- cated and the cytosol obtained by ultracentrifugation at 104,000g for 1 h. (A} Low molecular mass standards (LKB, Pharmacia, Sweden); (B) without sodium selenite; (C) 1.0 ~tM sodium selenite.


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182 B r t k o et al.

centration of selenite remaining the GH4C1 cell vitality and growth remain unchanged. The data showing the prolactin secretion at 2.5 ~tM sodium selenite as unchanged suggest that the selenite at the above concentration has no effect on prolactin synthesis in GH4C1 cells. On the other hand, it may decrease the rate of TRc~I and TRc~2 mRNAs synthesis and/or increase the rate of degradation of the above mRNAs. Sodium selenite (1.0 WV/) also causes significant changes in both the electrophoretic profile of cytoplasmic proteins and their phosphorylation in GH4C1 cells. However, with the present data, it is not possible to explain the biological significance of changes in electrophoretic profile and/or phosphorylation of cytoplasmic proteins in GH4C1 cells caused by 1.0 WV/sodium selenite.

In conclusion, our results based on the in vitro experiments in GH4C1 cells suggest that selenite may inhibit the T3 receptor ~1 gene expression; and selenite affects the protein phosphorylation, i.e., it may take part in the processes of postranslational modification of protein(s). Further work on the effect of different forms of inorganic or organic selenium on hormone nuclear receptors is warranted.

ACKNOWLEDGMENTS

We acknowledge the generosity of Ronald M. Evans for providing us 500 basepair Pvu II fragment from the neuronal c-erbA cDNA and Ana Aranda for sending us the GH4C1 pituitary cell line. We also wish to acknowledge the technical assistance of Viera Sedl~kov~ and Anna Krup- kovs This work was supported, in part, by the Grant Agency for Science, grants no. 2/542/93 and no. 2/999431/92.

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S o d i u m Se len i te a n d Ra t Pitui tary 183

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In vitro effects of sodium selenite on nuclear 3,5,3'-triiodothyronine (T3) receptor gene expression...

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