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Endocrinology Vol. 147, No. 12 5845-5854
Copyright © 2006 by The Endocrine Society

Regulation of Type III Iodothyronine Deiodinase Expression in Human Cell Lines

Monique H. A. Kester, George G. J. M. Kuiper, Rogier Versteeg and Theo J. Visser

Department of Internal Medicine (M.H.A.K., G.G.J.M.K., T.J.V.), Erasmus Medical Center, 3015 GE Rotterdam, The Netherlands; and Department of Human Genetics (R.V.), Academic Medical Center, Amsterdam, The Netherlands

Address all correspondence and requests for reprints to: Theo J. Visser, Department of Internal Medicine, Erasmus Medical Center, Room Ee 502, Dr Molewaterplein 50, 3015 GE, Rotterdam, The Netherlands. E-mail: t.j.visser{at}erasmusmc.nl.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Type I iodothyronine deiodinase (D1) and type II iodothyronine deiodinase (D2) catalyze the activation of the prohormone T4 to the active hormone T3; type III iodothyronine deiodinase (D3) catalyzes the inactivation of T4 and T3. D3 is highly expressed in brain, placenta, pregnant uterus, and fetal tissues and plays an important role in regulating thyroid hormone bioavailability during fetal development. We examined the activity of the different deiodinases in human cell lines and investigated the regulation of D3 activity and mRNA expression in these cell lines, as well as its possible coexpression with neighboring genes Dlk1 and Dio3os, which may also be especially important during development. D1 activity and mRNA were only found in HepG2 hepatocarcinoma cells, and D2 activity was observed in none of the cell lines. D3 activity and mRNA was found in ECC-1 endometrium carcinoma cells, MCF-7 mammacarcinoma cells, WRL-68 embryonic liver cells, and SH-SY5Y neuroblastoma cells, but not in the HepG2 hepatocarcinoma cell line or in any choriocarcinoma or astrocytoma cell line. We demonstrated that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate increased D3 activity 2- to 9-fold in ECC-1, MCF-7, WRL-68, and SH-SY5Y cells. Estradiol increased D3 activity 3-fold in ECC-1, but not in any other cells. Dexamethasone decreased D3 activity in WRL-68 cells only in the absence of fetal calf serum. Incubation with retinoids increased D3 activity 2- to 3-fold in ECC-1, WRL-68, and MCF-7 cells but decreased D3 activity in SH-SY5Y cells. D3 expression in the different cells was not affected by cAMP or thyroid hormone. Interestingly, D3 mRNA expression in the different cell lines strongly correlated with Dio3os mRNA expression and in a large set of neuroblastoma cell lines also with Dlk1 expression. In conclusion, we identified different human D3-expressing cell lines, in which the regulation of D3 expression is cell type-specific. Our data suggest that estradiol may be one of the factors contributing to the induction of D3 activity in the pregnant uterus and that in addition to gene-specific regulatory elements, more distant common regulatory elements also may be involved in the regulation of D3 expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE BIOAVAILABILITY OF thyroid hormone is regulated by three iodothyronine deiodinases [type I iodothyronine deiodinase (D1), type II iodothyronine deiodinase (D2), and type III iodothyronine deiodinase (D3)]. D1 and D2 catalyze the outer ring deiodination of the prohormone T4 to the receptor-active T3 (1). D1 is expressed in liver, kidney, thyroid, and pituitary (1). In liver and kidney, D1 is positively regulated by T3 (2). In thyroid, D1 expression is stimulated by T3, TSH, and TSH receptor-stimulating antibodies (3, 4). D2 is primarily expressed in brain, pituitary, and brown adipose tissue (1). In general, D2 is negatively regulated by thyroid hormone and positively regulated via cAMP (1).

D3 catalyzes the inner ring deiodination of T3 to 3,3'-diiodothyronine and of T4 to reverse T3 (rT3). In the adult, D3 is predominantly expressed in brain and skin (5, 6). Much higher D3 expression has been demonstrated in various fetal tissues such as liver and brain and at sites of the maternal-fetal interface, such as placenta, uterus, and umbilical arteries and vein (7, 8, 9, 10, 11, 12). The high expression in the fetal compartment suggests that D3 plays an important role in the regulation of fetal circulating T3 levels as a mechanism to protect the fetus from undue exposure to active thyroid hormone. This key function of D3 during development is also demonstrated by the growth retardation, neonatal mortality, and abnormal thyroid status of D3 knockout mice (13).

Besides its role during fetal development, D3 may have a pathophysiological role during illness. It has been demonstrated recently that severe hypothyroidism is induced by high levels of D3 expression in vascular tumors, a condition that is referred to as consumptive hypothyroidism (14, 15). Also, in nonthyroidal illness, increased D3 expression may contribute to the decreased serum T3 and the increased serum rT3. This concept is supported by a study by Peeters et al. (16) on deiodinase expression and serum thyroid hormone parameters in critically ill patients. In these patients, besides a down-regulation of hepatic D1, an elevated D3 expression was found in liver and skeletal muscle.

Different in vivo studies have been performed to investigate potential regulatory effects of different factors on D3 expression. In pig cerebrum, D3 activity is positively regulated by thyroid hormone (17). D3 activity is also under positive control of thyroid state in rat brain and skin (6, 18, 19), whereas D3 activity in the rat placenta is unaffected by hyper- and hypothyroidism (20). In the chicken, Van der Geyten et al. (21, 22) demonstrated an acute pretranslational down-regulation of D3 by dexamethasone (DEX) and growth hormone in the embryonic liver, but not in the brain.

To further clarify the role of D3 in thyroid hormone homeostasis, several studies have been performed on the regulation of its expression in different rat primary cultures. D3 activity was found to be induced in cultured rat brown adipocytes and brown fat vascular stromal cells by 12-O-tetradecanoylphorbol-13-acetate (TPA) and by several growth factors, such as epidermal growth factor (EGF) and fibroblast growth factor (FGF) (23, 24, 25), and in cultured astroglial cells by T3, retinoids, cAMP, TPA, and growth factors (26, 27, 28). In various human primary cultures including fibroblasts, skeletal muscle myoblasts, and endothelial cells, D3 has been shown recently to be increased by TGFß via a MAPK-dependent pathway (29). This TGFß stimulation synergized with the induction of D3 by TPA, EGF, and FGF (29).

Interestingly, recent literature shows that the Dio3 gene is subject to imprinting, with preferential expression from the paternal allele (30, 31). The gene is part of a large cluster of imprinted genes located on mouse chromosome 12F1 and human chromosome 14q32. Other genes in this locus include Dio3os, which is a nonprotein coding gene that is transcribed antisense to Dio3 (32), and the paternally expressed Dlk1 gene, which is a member of the EGF-like protein family and plays a role in growth and differentiation of several tissues (33). Recent microarray data show that the imprinted locus of human chromosome 14 is subject to parental allele and also tissue-specific expression (34), which suggests coregulation of the different imprinted genes on chromosome 14q32.

In the present study, we investigated the regulation of D3 expression in different human cell lines to further understand the role of D3 in the regulation of thyroid hormone homeostasis. We screened different human cell lines, which were used as models for D3-expressing tissues, for deiodinase activities and mRNA expression and determined the functionality and tissue specificity of potential regulatory factors concerning D3 expression. Furthermore, to investigate if in addition to gene-specific more distant common regulatory elements also may be involved in the regulation of D3 expression, we studied the possible coregulation of Dio3, Dio3os, and Dlk1 by determining the Dio3, Dio3os, and Dlk1 mRNA expression in these cell lines.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
[3'-125I]T3 and [3',5'-125I]T4 were obtained from GE Healthcare, Little Chalfont (Buckinghamshire, UK), and [3',5'-125I]rT3 was prepared by radioiodination of 3,3'-diiodothyronine as described previously (35). Nonradioactive iodothyronines were purchased from Henning Berlin GmbH (Berlin, Germany); 8-bromo-cAMP (8-Br-cAMP), TPA, DEX, 17ß-estradiol (E2), progesterone (P), 9-cis-retinoic acid (RA), human chorionic gonadotrophin, and 6-n-propyl-2-thiouracil (PTU) from Sigma (St. Louis, MO); all-trans-RA (tRA) from Janssen-Cilag (Tilburg, The Netherlands); the progestin R5020 from Perkin-Elmer (Wellesley, MA); dithiothreitol (DTT) from ICN Biochemicals Inc. (Costa Mesa, CA); and Sephadex LH-20 from Pharmacia (Woerden, The Netherlands). The RA receptor (RAR) pan-agonist CD367 and the retinoid X receptor (RXR) agonist CD3640 were kindly provided by Prof. Dr. U. Reichert (Galderma Research and Development, Sophia Antipolis, France). Real-time PCR probes and primers were obtained from Biosource (Nivelles, Belgium), and synthetic oligonucleotides for semiquantitative PCR were obtained from Life Technologies, Inc. (Gaithersburg, MD).

Response element (RE) luciferase constructs
The RE luciferase plasmids were constructed as previously described. The estrogen-responsive reporter gene construct (3xERE-TATA-luc) contains three copies of a consensus estrogen RE (ERE) and a TATA box in front of the luciferase gene (36). The glucocorticoid RE-containing mouse mammary tumor virus (MMTV) promoter-luciferase (luc) construct was kindly provided by Organon (Oss, The Netherlands). The TPA RE-luciferase construct (5xTPA-RE-TATA-luc) (37) was a gift from Dr. L. J. Blok (Erasmus Medical Center, Rotterdam, The Netherlands).

Cell culture
The different human cell lines were selected as models for D3-expressing tissues. The endometrium carcinoma cell line ECC-1 was kindly provided by Dr. B. van der Burg (Hubrecht Laboratory, Utrecht, The Netherlands), the mammary carcinoma cell line MCF-7 was kindly provided by Dr. J.A. Foekens (Department of Endocrine Oncology, Erasmus Medical Center), the Ishikawa endometrium carcinoma cell line IK-3H12 was provided by Dr. M. Nishida (Department of Obstetrics and Gynecology, University of Tsukuba, Tsukuba, Japan), and the choriocarcinoma cell line JAR was provided by Dr. C. Ris-Stalpers (Department of Pediatric Endocrinology, Amsterdam Medical Center, Amsterdam, The Netherlands). The neuroblastoma cell line SH-SY5Y, astrocytoma cell lines U87, U373, and CCF-STTG1, the embryonic liver cell line WRL-68, and the choriocarcinoma cell lines JEG-3 and BeWo were obtained from the European Collection of Cell Cultures (Salisbury, UK). All cell lines were grown in DMEM/Ham’s F-12 medium (Life Technologies, Inc.), containing 9% fetal calf serum (FCS) and 100 nM sodium selenite. At confluency, cells were split and seeded in six- or 12-well culture dishes in DMEM/F12, containing 7% FCS or 7% charcoal-treated (CT) FCS; 24 h after seeding, incubations were started. Incubations with E2 and/or P were done in medium without phenol red. Cells were incubated with 1–10 nM E2 (24–48 h), 10–100 nM P (24–48 h), 10–1000 nM DEX (1–24 h), 3 µM tRA, 9-cis-RA, CD367, or CD3640 (30–48 h), or 0.1 µM TPA (6 h).

Deiodinase activity assays
Cells were incubated under different conditions in six- or 12-well culture dishes. After incubation, the cells were rinsed twice with PBS and harvested by scraping the content of each well into 100 mM phosphate buffer containing 2 mM EDTA and 1 mM DTT and disrupted by sonication. The cell sonicates were stored at –80 C until further analysis. Protein content was determined using the method of Bradford (38), with BSA as standard.

D1 activities were determined by incubation of 0.1 µM [3',5'-125I]rT3 (100,000 cpm) for 120 min at 37 C with 0.2–1 mg protein/ml cell sonicate in the presence or absence of 0.1 mM PTU in 0.1 ml 0.1 M phosphate (pH 7.2), 2 mM EDTA, and 10 mM DTT (PED10). Reactions were stopped by the addition of 0.1 ml 5% BSA. Protein-bound [125I]iodothyronines were precipitated by addition of 0.5 ml 10% trichloroacetic acid. After centrifugation, the supernatants were analyzed for 125I– production on Sephadex LH-20 minicolumns (bed volume, 0.25 ml), equilibrated, and eluted with 0.1 M HCl. The difference between incubations with and without PTU represents D1 activity.

D2 activities were determined by incubation of 1 nM [3',5'-125I]T4 (100,000 cpm) for 120 min at 37 C with 0.2–1 mg/ml cell sonicate in the presence of 100 nM unlabeled T3 to inhibit D3 activity and in the absence or presence of 100 nM T4 to saturate D2 in 0.1 ml PED10. Release of 125I– was determined as described for D1 activity. The difference between incubations with 1 and 100 nM T4 represents D2 activity.

D3 activities were measured by incubation of 1 nM [3'-125I]T3 (200,000 cpm) for 60–240 min at 37 C with 0.05–1 mg protein/ml cell sonicate in 0.1 ml PED10 (pH 7.2). To validate the D3 assay, some incubations were also carried out in the presence of a D3-saturating concentration (100 nM) unlabeled T3. Reactions were stopped by the addition of 0.1 ml ice-cold methanol. After centrifugation, 0.15 ml supernatant was mixed with 0.1 ml 0.02 M ammonium acetate (pH 4.0), and 0.1 ml of the mixture was applied to a 4.6- x 250-mm Symmetry C18 column connected to an Alliance HPLC system (Waters, Etten-Leur, The Netherlands) and eluted with a gradient of acetonitrile in 0.02 M ammonium acetate (pH 4.0) at a flow of 1.2 ml/min. The proportion of acetonitrile was increased linearly from 28–42% in 15 min. The radioactivity in the eluate was determined using a Radiomatic A-500 flow scintillation detector (Packard, Meriden, CT).

Reporter luciferase assay
Twenty-four hours after transfection, 10 nM E2 or DEX or 100 nM TPA was added. Twenty-four (E2, T3, DEX) or 6 (TPA) h later, cells were lysed in 150 µl lysis buffer [25 mM Tris phosphate (pH 7.8), 15% glycerol, 1% Triton X-100, 1 mM DTT, and 8 mM MgCl2]. Luciferase activity was measured in 25 µl lysate in a TOPCOUNT luminometer (Applied Biosystems, Nieuwerkerk a/d IJssel, The Netherlands), using the Dual Glo luciferase assay system (Promega, Madison, WI). Luminescence measured from the pCMV-Renilla or pSV40-Renilla was used to correct for transfection efficiency.

RNA isolation and RT
After incubation, RNA was isolated from 106 cells of the above-mentioned cell lines using the high pure RNA isolation kit (Roche Diagnostics, Almere, The Netherlands) according to the manufacturer’s guidelines. RNA concentrations were determined using RiboGreen RNA quantitation kit (Molecular Probes, Leiden, The Netherlands). RNA (500 ng) was used for cDNA synthesis using TaqMan RT reagents (Roche Diagnostics). RNA samples were verified to be free from genomic DNA by performing negative control cDNA synthesis reactions of 500 ng total RNA using Taqman RT reagents without reverse transcriptase.

RNA from 18 different neuroblastoma cell lines, which were previously analyzed for Dlk1 expression (39), was isolated using Trizol (Invitrogen, Breda, The Netherlands). For further purification, mRNA was isolated from 1 µg total RNA using the Dynabeads mRNA Purification Kit (Dynal Biotech, Hamburg, Germany). All isolated mRNA was used for cDNA synthesis using Taqman RT reagents.

RT-PCR
cDNA (2 µl) was used for semiquantitative and real-time PCR. The GeneAmp 9700 (Perkin-Elmer Inc., Torrance, CA) was used for semiquantitative PCR, and the ABI PRISM 7700 sequence detection system (Applied Biosystems) was used for real-time PCR. Table 1Go shows the sequences of the different primers and probes for real-time PCR and the synthetic oligonucleotides for semiquantitative PCR. mRNA levels are expressed relative to mRNA levels of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). GAPDH primers and probe for real-time PCR were provided as a preoptimized control system (Applied Biosystems).


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  		TABLE 1. Oligonucleotide primers used for semiquantitative and real-time PCR

 
Semiquantitative PCR was performed by comparing band intensities derived from target cDNAs with those obtained from GAPDH mRNA during the exponential phase of the reaction. Thirty-five reaction cycles were used for the PCR of D3 and Dio3os, 32 cycles for the PCR of Dlk1, and 27 cycles for the PCR of GAPDH. Annealing temperatures were 60 C for the PCR of D3, 50 C for the PCR of Dio3os and GAPDH, and 55 C for the PCR of Dlk1.

Real-time PCRs were done for 2 min at 50 C and for 10 min at 95 C, followed by 40 cycles of 15 sec at 95 C and for 1 min at 60 C. Per reaction, 200 nM D3 primers and probe or 300 nM Dlk1 primers and probe were used. Threshold cycle (Ct) values represent the cycle numbers at which probe-derived absorbance reaches the calculated threshold value. Data are expressed as {Delta}Ct (i.e. the Ct value of the housekeeping gene minus the Ct value of the target gene) or as 2{Delta}Ct x 1000 (relative number of mRNA copies).

Statistics
Results are given as means ± SEM or means ± range of values. Significance of differences between means was tested using the general linear model procedure (ANOVA) of the SPSS 10.1 statistical package (SPSS, Inc., Chicago, IL). P values ≤ 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
D3 activity in ECC-1 and WRL-68 cells
Because D3 is a selenoenzyme, we first tested the effect of selenite addition on D3 activity in ECC-1 cells. These cells were found to contain marked D3 activity. After culturing ECC-1 cells in selenite-deplete medium for 1 week, increasing concentrations of selenite were added to the medium for 96 h. Repletion of the medium with 10–100 nM selenite increased D3 activity 2–4-fold, respectively (Fig. 1Go). For this reason, in all further experiments, cells were cultured in medium containing 100 nM selenite.


Figure 1
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  		FIG. 1. Effect of selenite on D3 activity in ECC-1 cells. Cells were incubated for 96 h in the presence of 10–100 nM selenite. Reaction conditions for the D3 assay were 1 nM 125I-labeled T3, 0.3 mg protein/ml cell sonicate, and 3 h incubation. Results are the means ± range of values of triplicate determinations from a representative experiment.

 
E2 and P are known to increase D3 activity in uteri of ovariectomized rats (40). Furthermore, cAMP, T3, retinoids, and TPA are known to induce D3 activity, at least in astroglial cells (26, 27, 28), and DEX decreases D3 activity, at least in the chicken embryonic liver and in rat brown fat vascular stromal cells (21, 25). We first tested the responsiveness of ECC-1 and WRL-68 cells to most of these compounds by analyzing reporter activity in the presence or absence of the compounds in cells transfected with responsive element-luciferase reporter constructs. When ECC-1 cells were transfected with 3xERE-TATA-luc in medium with 7% CT-FCS, luciferase activity was 5-fold increased in the cells incubated with E2 (Fig. 2AGo). Seven percent nonstripped FCS increased luciferase activity 2.5-fold compared with 7% CT-FCS, masking the response of the cells to E2 (Fig. 2AGo). The responsiveness of ECC-1 cells to TPA was demonstrated by the 7-fold increase by TPA in 5xTPA-RE-TATA-luc transfected ECC-1 cells (Fig. 2BGo). In MMTV promoter-luc transfected WRL-68 cells, luciferase activity was increased more than 2-fold by DEX (Fig. 2CGo).


Figure 2
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  		FIG. 2. Effect of E2 on 3xERE-TATA-luc activity (A), TPA on 5xTPA-RE-TATA-luc activity (B), and DEX on MMTV promoter-luc activity (C) in ECC-1 (A and B) or WRL-68 (C) cells. Twenty-four hours after transfection with the different responsive element-luciferase constructs, cells were incubated with 100 nM E2 or 100 nM DEX for 24 h or with 100 nM TPA for 6 h in medium with the indicated serum. Results are the means ± range of values of duplicate determinations from a representative experiment. Significance of differences is indicated as follows: *, P < 0.05 for indicated differences.

 
Next, we investigated the effect of the different compounds on D3 activity in ECC-1 cells and found a 9-fold increase of D3 activity by a 6-h incubation with TPA, a 3-fold increase of D3 activity by a 48-h incubation with 9-cis or tRA, and no effect of an 8-h incubation with dibutyryl cAMP [(Bt)2-cAMP] and 8-Br-cAMP (Fig. 3AGo). Figure 3BGo shows that a 2–24-h incubation with cAMP did not affect D3 activity in ECC-1 cells. D3 activity was slightly increased by TPA after a 2-h incubation; the increase in D3 activity was maximal from 4–16-h incubation with TPA (Fig. 3BGo).


Figure 3
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  		FIG. 3. A, Effect of TPA, tRA, 9-cis-RA, and cAMP on D3 activity in ECC-1 cells. Cells were incubated with 0.1 µM TPA for 6 h, with 3 µM tRA or 3 µM 9-cis-RA for 48 h, or with 1 mM (Bt)2cAMP or 8-Br-cAMP for 8 h. Significance of differences is indicated as follows: *, P < 0.05 vs. control (–). B, Time course of effect of TPA and cAMP on D3 activity in ECC-1 cells. Cells were incubated with 0.1 µM TPA or with 1 mM (Bt)2-cAMP for 2–24 h. Reaction conditions for the D3 assay were 1 nM 125I-labeled T3, 0.3 (A) or 0.5 (B) mg protein/ml cell sonicate, and 2 h incubation. Results are the means ± range of values of duplicate determinations from a representative experiment.

 
Subsequently, we tried to mimic conditions during pregnancy by incubating the ECC-1 cells with E2 and P. Although P did not significantly affect D3 activity (Fig. 4AGo) or mRNA expression (Fig. 4BGo), in the absence of serum or in the presence of CT-FCS, E2 stimulated D3 activity and mRNA expression 3-fold. Nonstripped FCS increased D3 activity 5-fold. This increase may be partly due to the E2 present in the FCS (15 pM E2 in medium with 7% FCS) because no additional effect of E2 was found in this condition (Fig. 4Go). The effect of E2 on D3 activity seems to be cell type specific because no effect of E2 was found in WRL-68 cells (data not shown).


Figure 4
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  		FIG. 4. Effect of E2 and P on D3 activity and D3 mRNA levels in ECC-1 cells. Cells were incubated for 24 h with 100 nM E2 and/or P in the absence or presence of 7% CT serum or FCS. Reaction conditions for the D3 assay were 1 nM 125I-labeled T3, 0.15 mg protein/ml cell sonicate and 3 h incubation. Results are the means ± range of values of duplicate determinations from a representative experiment. Significance of differences is indicated as follows: *, P < 0.05 for indicated differences.

 
DEX down-regulates D3 expression in the embryonic chicken liver (21). We therefore also studied the effect of DEX on D3 activity in WRL-68 embryonic liver cells. Figure 5Go shows that DEX decreased D3 activity only in the absence of serum. Because T3 has been reported to up-regulate D3 expression in different tissues, we also investigated the possible effect of T3 on D3 activity in WRL-68 and ECC-1 cells. However, no effect of a 24–48-h incubation with 10 nM T3 was found in these cells (results not shown). Although mRNA of the thyroid hormone receptors {alpha}1, {alpha}2, and ß1 was present in these cells, transfection studies on thyroid hormone responsiveness of the different cells remained inconclusive because not only the 2xTRE Firefly luciferase was up-regulated by thyroid hormone but also the control SV40 Renilla luciferase (data not shown).


Figure 5
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  		FIG. 5. Effect of DEX on D3 activity in WRL-68 cells. Cells were incubated for 24 h with 100 nM DEX. Reaction conditions for the D3 assay were 1 nM 125I-labeled T3, 0.15 mg protein/ml cell sonicate, and 3 h incubation. Results are the means ± SEM of three different experiments. Significance of differences is indicated as follows: *, P < 0.05 for indicated differences.

 
Effects of retinoids, TPA, and cAMP on deiodinase activities in different cell lines
Table 2Go compares deiodinase activities in 12 different cell lines. D2 activity was absent in all cell lines, D1 activity was only observed in HepG2 cells (Table 2Go), and no effect of cAMP or TPA on D2 or D1 activity was found. D3 activity was found in ECC-1 endometrium carcinoma cells, but not in Ishikawa endometrium carcinoma cells, and in WRL-68 embryonic liver cells, but not in the adult HepG2 hepatocarcinoma cell line. Furthermore, D3 activity was present in the neuroblastoma cell line SH-SY5Y and in the mammacarcinoma cell line MCF-7. D3 activity was not observed in any of the choriocarcinoma and astrocytoma cell lines tested (Table 2Go). Table 2Go also shows the effect of cAMP, tRA, and TPA on D3 activity in the different cell lines. cAMP did not affect D3 activity in any cell line. TPA increased D3 activity 9-fold in ECC-1 cells, 2-fold in WRL-68, 5-fold in MCF-7 cells, and 1.5-fold in SH-SY5Y cells. tRA increased D3 activity 3-fold in ECC-1 cells and 2-fold in WRL-68 and MCF-7 cells but decreased D3 activity 3-fold in SH-SY5Y cells (Table 2Go).


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  		TABLE 2. Deiodinase activities and folds increase in D3 activity in different human cell lines

 
Although all-trans- and 9-cis-RA signal through binding to both RARs and RXRs, CD367 is a specific RAR agonist, and CD3640 is a specific RXR-agonist. To verify whether the retinoids acted via RAR or RXR receptors, we tested the effects of different RAR and RXR ligands on D3 activity in ECC-1, SH-SY5Y, and WRL-68 cells. Although all RAR and RXR ligands induced D3 activity in ECC-1 cells, they diminished D3 activity in SH-SY5Y cells (Fig. 6Go). Figure 7Go shows D3 activity and mRNA expression in ECC-1 and WRL-68 cells. In these cell lines, all retinoids up-regulated both D3 mRNA expression and D3 activity, indicating that the regulation is largely at the pretranslational level.


Figure 6
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  		FIG. 6. Effect of RAR and RXR ligands on D3 activity in ECC-1 and SH-SY5Y cells. Cells were incubated for 48 h with 0.3–10 µM tRA, 9-cis-RA, CD367, or CD3640. Reaction conditions for the D3 assay were 1 nM 125I-labeled T3, 0.5 mg protein/ml cell sonicate, and 2 h incubation. Results are the means ± range of values of duplicate determinations from a representative experiment. P values refer to trend analysis.

 

Figure 7
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  		FIG. 7. Effect of different RAR and RXR receptor ligands on D3 activity and D3 mRNA level in ECC-1 and WRL-68 cells. Cells were incubated for 48 h with 3 µM tRA, 9-cis-RA, CD367, or CD3640. Reaction conditions for the D3 assay were 1 nM 125I-labeled T3, 0.5 mg protein/ml cell sonicate, and 2 h incubation. Results are the means ± range of values of duplicate determinations from a representative experiment. Significance of differences is indicated as follows: *, P < 0.05 vs. control (–).

 
Coexpression of genes of the imprinted region on chromosome 14q32
Figure 8Go depicts the mRNA expression of D3 and its neighboring genes hDio3os and Dlk1. The designated bands represent specific bands of the expected length (552 bp for hD3, 518 bp for Dlk1, 941 bp for GAPDH, and 1470 and 657 bp for two different transcripts of Dio3os). Except for Ishikawa cells, in which despite the absence of D3 activity, a very low level of D3 mRNA was detected, we found that D3 mRNA and hDio3os mRNA were present in the same cell lines in which D3 activity is present, i.e. in ECC-1, WRL-68, SH-SY5Y, and MCF-7 cells. In contrast to D3 and Dio3os, no significant Dlk1 expression was found in ECC-1, WRL-68, and MCF-7 cells. Dlk1 mRNA was clearly found to be abundantly expressed in HepG2 hepatocarcinoma cells, which lack D3 and Dio3os, and in SH-SY5Y neuroblastoma cells, which also express D3 and Dio3os.


Figure 8
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  		FIG. 8. RT-PCR of D3, Dio3os, Dlk1, and GAPDH mRNA in different cell lines. 1, ECC-1; 2, ECC-1 incubated for 4 h with 0.1 µM TPA; 3, WRL-68; 4, HepG2; 5, Jeg3; 6, JAR; 7, BeWo; 8, U87; 9, U373; 10, CCF-STTG1; 11, SH-SY5Y; 12, MCF-7; 13, Ishikawa; 14, COS-1.

 
We also studied the possible coexpression of the different genes in 18 different neuroblastoma cell lines, first by semiquantitative PCR, which showed a positive correlation of D3 with Dio3os and Dlk1 (Fig. 9AGo). We investigated the expression of D3 and Dlk1 further by quantitative PCR and confirmed a positive correlation between both genes in these cell lines (r = 0.797, Fig. 9BGo).


Figure 9
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  		FIG. 9. Expression of D3, Dio3os, Dlk1, and GAPDH mRNA (RT-PCR, A) and correlation between Dlk1 and D3 mRNA expression (Q-PCR, B) in different human neuroblastoma cell lines. 1, SKNFi; 2, N206; 3, KCNR; 4, SKNAS; 5, Lan-1; 6, Lan-5; 7, Lan-6; 8, SKNSH; 9, Tet2; 10, Tet21N; 11, SJNB-1; 12, SJNB-6; 13, SJNB-8; 14, SJNB-10; 15, NMB; 16, SKNBe; 17, IMR32; 18, N110B.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Studies of the regulation of D3 expression in cultured cells contribute to our understanding of the role of D3 in the regulation of thyroid hormone homeostasis. In previous studies, D3 was found to be expressed in primary cultures of rat astrocytes and brown preadipocytes (23, 26). The regulation of D3 expression was studied in these primary cultures or in the BVS-1 cell line, which is derived from the vascular stromal fraction of rat neonatal brown fat (25). In these rat cell culture models, TPA, EGF and FGF, cAMP, T3, and retinoids were identified as factors inducing D3 activity (23, 26). In addition, with respect to human cell cultures, endogenous D3 expression has been found in the Caco-2 colon carcinoma cell line (41), and recently, Huang et al. (29) showed D3 expression in primary cultures of fibroblasts, hemangioma cells, endothelial cells, and skeletal muscle myoblasts and identified TGF-ß as a factor inducing D3 activity in these cultures. In this study, we tested a panel of human cell lines for endogenous D3 activity and used the cell lines expressing D3 as models for human D3-expressing tissues to study the effect of different regulatory factors on D3 expression.

Previous immunolocalization experiments have demonstrated placental D3 in the syncytiotrophoblasts and cytotrophoblasts and in the fetal endothelium in chorionic villi and the maternal decidua (11). First, we investigated D3 expression in different human choriocarcinoma cell lines. Even though these cell lines were derived from trophoblasts, no D3 expression was found in any choriocarcinoma cell line, indicating that these cell lines cannot be used as models for placental D3-expressing trophoblasts.

Consistent with the expression of D3 in uterine endometrial glands (11), endogenous D3 mRNA expression and D3 activity was detected in ECC-1 endometrium carcinoma cells, and low but significant D3 mRNA was detected in Ishikawa endometrium carcinoma cells. In the rat uterus, D3 activity is highly induced during pregnancy, especially during the implantation process (12). Because E2 and P are important for the implantation process and synergistically increase uterine D3 expression in ovariectomized rats (40), these steroid hormones are likely to contribute to the regulation of uterine D3 expression. In agreement with this, we found a positive effect of E2 on D3 activity and mRNA expression in ECC-1 endometrium carcinoma cells but not in other cells, suggesting a cell type-specific regulation at the transcriptional level.

TPA is a phorbol ester, which activates the downstream Raf/MEK/Erk cascade via protein kinase C (PKC) (42, 43, 44). Johnson et al. (45) demonstrated that TPA can mimic E2 and EGF for initiating implantation, and Chen et al. (46) showed that the PKC pathway is induced in the uterine epithelium during implantation by the synergistic action of P and E2, suggesting that this pathway plays an important role in modulating steroid hormone responsiveness in the uterine luminal epithelium during implantation. Therefore, we also investigated the effect of TPA on D3 activity in ECC-1 cells and confirmed a PKC-mediated increase of D3 expression in these cells.

In addition to D3 expression in endometrium carcinoma cells, D3 mRNA expression and activity were also found in neuroblastoma cells, but not in the different astrocytoma cell lines tested. This is in agreement with in situ hybridization data by Escamez et al. (47), who showed D3 expression in neuronal cells but not in astrocytes. Our findings of D3 activity and mRNA expression in WRL-68 embryonic liver cells, but not in HepG2 hepatocarcinoma cells, and of D1 activity in HepG2, but not in WRL-68 cells, are consistent with D3 being present in fetal, but not in adult human liver, and with D1 expression being higher in adult than in embryonic human liver (8). Furthermore, the presence of D3 activity in MCF-7 mammacarcinoma cells might indicate that D3 is expressed in breast tissue under certain conditions, such as during the lactation period. However, this remains to be investigated.

DEX and thyroid hormone have also been reported to affect D3 expression. DEX was shown to acutely down-regulate D3 expression in hepatic chicken liver (21), and in vitro studies demonstrated a DEX-mediated decrease of basal and growth factor-induced D3 activity in BVS-1 cells (25). In our study, in line with these findings, DEX decreased D3 activity in WRL-68 embryonic liver cells in medium without serum. In pig and rat brain and rat skin, D3 is increased in hyperthyroidism and decreased in hypothyroidism (6, 17, 18). In addition, thyroid hormone was shown to induce D3 activity in astroglial cells (27). In contrast to these findings, thyroid hormone did not regulate D3 activity in the cell lines tested in our study, despite the presence of mRNA coding for the thyroid hormone receptors {alpha}1, {alpha}2, and ß1.

The second messenger, cAMP, is known to dramatically induce expression of D2, and a functional cAMP RE has been identified in its gene promoter (48, 49). Neither under basal conditions nor after incubation with cAMP did any of the cell lines tested in the present study express D2 activity. We focused on the regulation of D3 expression and analyzed the possible involvement of the PKA pathway in this regulation. In our model of different cell lines, the PKA pathway seems not to be involved in the regulation of D3 expression because no effect of cAMP was found in any cell line.

Retinoids are vitamin A derivatives that regulate cellular growth and differentiation (50). RA mediates its diverse effects via two families of nuclear hormone receptors, which both have three different isoforms: RARs and RXRs {alpha}, ß, and {gamma}. tRA exerts its effects via the RARs, and 9-cis-RA acts through both RARs and RXRs but has higher affinity for the RXRs. RXRs form heterodimers with RARs but also with other nuclear hormone receptors such as thyroid hormone receptors and bind as heterodimers to specific REs of target genes. RARs and RXRs recognize a much wider range of REs than e.g. the estrogen receptor (51), which may account for the diversity of the effects mediated by retinoids. Previously, retinoids have been reported to down-regulate TSHß mRNA levels and promoter activity (52, 53) and D2 mRNA levels, to up-regulate D1 activity in liver, pituitary, and human follicular thyroid carcinoma cell lines (52, 54), and to up-regulate D3 expression in astroglial cells (28). In the present study, we demonstrated that the effects of retinoids on D3 expression are dependent on the cell-type. D3 expression was up-regulated by the RAR and RXR ligands in ECC-1 and WRL-68 cells but down-regulated in the SH-SY5Y cells. Because within a cell line all RAR and RXR ligands showed similar effects, in all retinoid-responsive cell lines, both RAR and RXR pathways seem to be involved in the regulation of D3 expression. The receptors may regulate D3 activity directly by binding to a RA responsive element or RXresponsive element in the D3 promoter. No such RE has been identified in the D3 promoter yet. Alternatively, the receptors act on cell type-specific RA-responsive genes that up- or down-regulate D3 expression.

The Dio3 gene is located on human chromosome 14q32 and is imprinted such that the paternal allele is preferentially expressed (30). Dlk1 and Dio3os are located in the same imprinted region as Dio3. Like D3, Dlk1 is abundantly expressed in fetal tissues. We studied the expression of D3, Dlk1, and Dio3 mRNA in different cell lines to investigate whether besides imprinting, tissue-specific and pathophysiological regulation of D3 expression may also be associated with Dio3os and Dlk1 expression. In the panel of different human cell lines, there was a good correlation between D3 and Dio3 mRNA expression, but no correlation was found between D3 and Dlk1 mRNA expression. In HepG2 cells, Dlk1 expression was high, whereas D3 and Dio3os expression were absent, indicating that the regulatory elements involved in expression of Dlk1 in HepG2 cells are mainly gene-specific.

Many neuroblastomas arise in the adrenal medulla, which primarily consists of chromaffin cells. Precursor neuroblasts can develop into sympathetic neurons or chromaffin cells. In the set of different neuroblastoma cell lines, which represent different developmental stages of neuroblast development (39), we did find coexpression of Dlk1 and D3. This suggests that common regulatory elements are involved in the expression of these genes in these different cell lines and thus at specific neuroblast differentiation stages. The highest Dlk1 and D3 mRNA expression was found in the cell lines Lan-6, SJNB-1, and SKNBe, which are all of the chromaffin differentiation lineage (39).

In summary, we identified a series of D3-expressing human cell lines, which were used as models for D3-expressing tissues, to investigate the regulation of D3 expression. We demonstrated that induction of PKC leads to a large increase of D3 activity in endometrium carcinoma cells and that E2 up-regulates D3 expression in these cells. These findings suggest that the PKC pathway may be an important pathway involved in the induction of D3 observed in the pregnant rat uterus during the implantation process and that E2 may be one of the factors contributing to this induction of uterine D3. Furthermore, we showed that the regulation of D3 expression by retinoids involves both RAR and RXR pathways and is cell type-specific. In addition, we showed that D3 and Dio3os are coexpressed in our set of human cell lines and that D3, Dio3os, and Dlk1 are coexpressed in a set of different human neuroblastoma cell lines, suggesting that besides being gene-specific, also more distant common regulatory elements are involved in the regulation of the expression of the different genes. Further characterization of the molecular mechanisms involved in the regulation of D3 expression will help to unravel the role of D3 in the regulation of thyroid hormone bioavailability during fetal development and during illness.


   Acknowledgments
 
We thank Dr. B. van der Burg for the generous gift of ECC-1 cells, Dr. M. Nishida for the gift of the Ishikawa cells IK-3H12, and Dr. C. Ris-Stalpers for providing the JAR cells. We also thank Organon for providing the MMTV promoter-luc construct, Dr. L. J. Blok for the gift of 5xTPA-RE-TATA-luc, and Prof. Dr. U. Reichert for kindly providing the RAR and RXR agonists CD367 and CD3640.


   Footnotes
 
This work was supported by the European Community (Grant QLG-2000-00930) and by The Netherlands Organization for Scientific Research (Grants 903-40-204 and 916-56-186).

The authors have nothing to disclose.

First Published Online August 24, 2006

Abbreviations: 8-Br-cAMP, 8-Bromo-cAMP; (Bt)2-cAMP, dibutyryl cAMP; CT, charcoal treated; Ct, threshold cycle; D1, type I iodothyronine deiodinase; D2, type II iodothyronine deiodinase; D3, type III iodothyronine deiodinase; DEX, dexamethasone; DTT, dithiothreitol; E2, 17ß-estradiol; EGF, epidermal growth factor; ERE, estrogen RE; FCS, fetal calf serum; FGF, fibroblast growth factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; luc, luciferase; MMTV, mouse mammary tumor virus; P, progesterone; PED10, 0.1 M phosphate (pH 7.2), 2 mM EDTA and 10 mM DTT; PKC, protein kinase C; PTU, 6-n-propyl-2-thiouracil; RA, retinoic acid; RAR, retinoic acid receptor; RE, response element; rT3, reverse T3; RXR, retinoid X receptor; TPA, 12-O-tetradecanoylphorbol-13-acetate; tRA, all-trans-retinoic acid.

Received May 4, 2006.

Accepted for publication August 14, 2006.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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