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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2006-2358
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The Journal of Clinical Endocrinology & Metabolism Vol. 92, No. 10 4046-4051
Copyright © 2007 by The Endocrine Society

Effect of Iodide on Human Choriogonadotropin, Sodium-Iodide Symporter Expression, and Iodide Uptake in BeWo Choriocarcinoma Cells

Huika Li, Kerry Richard, Brett McKinnon and Robin H. Mortimer

Department of Endocrinology (H.L., K.R., B.M., R.H.M.), Conjoint Endocrine Laboratory, Royal Brisbane and Women’s Hospital and Queensland Health Pathology Services, Brisbane, Queensland 4029, Australia; and Discipline of Obstetrics and Gynaecology (R.H.M.), The University of Queensland, Brisbane, Queensland 4072, Australia

Address all correspondence and requests for reprints to: Dr. Robin Mortimer, Department of Endocrinology, Royal Brisbane, Women’s Hospital, Herston, Brisbane, Queensland 4029, Australia. E-mail: robin_mortimer{at}health.qld.gov.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Context: Active placental transport of maternal iodide by the thyroidal sodium iodide symporter (NIS) provides an essential substrate for fetal thyroid hormone synthesis. NIS is expressed in trophoblast and is regulated by human choriogonadotropin (hCG). In thyroid, iodide down-regulates expression of several genes including NIS. Placentas of iodine-deficient rats demonstrate up-regulation of NIS mRNA, suggesting a role for iodide in regulating placental NIS.

Objectives and Methods: The objectives were to examine effects of iodide on expression of NIS and hCG in BeWo choriocarcinoma cells. Gene expression was studied by quantitative real-time PCR. Effects on NIS protein expression were assessed by Western blotting. Functional activity of NIS was measured by 125I uptake. Expression of hCG protein was assessed by immunoassay of secreted hormone.

Results: Iodide inhibited NIS mRNA and membrane protein expression as well as 125I uptake, which were paralleled by decreased ßhCG mRNA expression and protein secretion. Iodide had no effects on pendrin expression. Addition of hCG increased NIS mRNA expression. This effect was partially inhibited by addition of iodide. The inhibitory effects of iodide on NIS mRNA expression were abolished by propylthiouracil and dithiothreitol.

Conclusions: We conclude that expression of placental NIS is modulated by maternal iodide. This may occur through modulation of hCG effects on NIS and hCG gene expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE FETAL THYROID begins to concentrate iodide, essential for thyroid hormone synthesis and secrete thyroid hormone by about 12–16 wk gestation (1). It is increasingly apparent that even a mild reduction in fetal thyroid hormone levels can lead to irreversible central nervous system damage to the child (2, 3, 4), and worldwide, iodine deficiency remains a major cause of neonatal mental retardation.

The placenta provides iodide to the fetus by active transport of maternal iodide and by deiodination of maternal thyroid hormone. Two iodide transporters have been identified in placenta, sodium/iodide symporter (NIS) and pendrin, a protein encoded by the Pendred syndrome gene (5, 6). Both have been previously reported in thyroid. In placenta and polarized human choriocarcinoma BeWo cells, NIS is located in the apical (maternal) membrane (6, 7). Our recently published physiological study (7) suggested that, as in thyroid tissue, BeWo cells accumulate iodide through NIS and release it through pendrin. In thyroid, NIS gene expression is up-regulated by TSH and the structurally homologous placental hormone, human choriogonadotropin (hCG) (8, 9). hCG also stimulates NIS expression and iodide uptake in choriocarcinoma (JAr) cells (10). There is persuasive evidence that iodide inhibits both NIS expression and iodide uptake in thyroid (11, 12, 13). Thyroid NIS mRNA is also up-regulated in fetuses of iodine-deficient rats (14), signifying a regulatory role for iodine in the fetal thyroid. Placental NIS mRNA expression is also up-regulated in these rats. This latter finding suggests that placental NIS expression and iodide uptake may also be directly or indirectly regulated by iodide.

We therefore investigated the effect of iodide on NIS, hCG expression, and iodide uptake in the human choriocarcinoma BeWo cell line as a model of human trophoblast. We found that iodide inhibits expression of NIS, hCG, and iodide uptake in these cells but has no effect on pendrin gene expression. Iodide also reduced hCG-stimulated NIS expression. Inhibition of NIS by iodide was restored by propylthiouracil (PTU) and dithiothreitol (DTT). We infer that placental iodide concentration regulates placental iodide transfer.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and treatments

Choriocarcinoma cells, BeWo (CCL 98, passage 195) were obtained from the American Type Culture Collection (Manassas, VA) and cultured in nutrient mixture F-12 (Ham) medium. Media were supplemented with 10% (vol/vol) fetal bovine serum (Invitrogen Lifetechnologies, Carlsbad, CA), 500 U/ml penicillin (Invitrogen), 500 U/ml streptomycin (Invitrogen), and 5 µg/ml plasmocin (Integrated Sciences, Chatswood, New South Wales, Australia). Cells were passaged twice a week and media changed on alternate days. All experiments were performed on cells between passage 12 and 18 from original stock. Viability of cells was estimated by Trypan blue exclusion and lactate dehydrogenase (LDH) release assay.

In experiments, BeWo cells were seeded in six-well plates for 2 d to reach confluence. Then cells were treated with 0, 1, 10, and 100 µM of NaI (Sigma-Aldrich, Castle Hill, New South Wales, Australia) in 10% fetal bovine serum-DMEM for 4 and 24 h for mRNA and 3 d for protein and iodide uptake. To examine the mechanism of iodide effects on NIS, the cells were incubated with NaI plus hCG (Organon, Lane Cove, Australia), PTU (Sigma), or DTT (Invitrogen).

Real-time quantitative PCR

Total cellular RNA was isolated from cells using the RNeasy minikit (QIAGEN, Doncaster, Victoria, Australia). RNA concentrations were determined spectrophotometrically, and RNA quality was assessed by agarose gel electrophoresis and A260 to A280 ratio. Four micrograms of total RNA were reverse transcribed with Superscript III reverse transcriptase and oligo (dT) 15 primers (Invitrogen Life Technologies) in a reaction volume of 20 µl.

Real-time quantitative PCR was performed using cDNA synthesized from 0.03 µg RNA, 20 µM of each primer, and SYBR green PCR master mix (AB Applied Biosystems, Warrington, UK) in a Rotor Gene RG-3000 (Corbett Research, Mortlake, New South Wales, Australia). Primer sequences were obtained from an online database (http://pga.mgh.harvard.edu/primerbank/index.html) and synthesized by Sigma-Genosys (Victoria, Australia). ß2-Microglobulin (ß2M) was used as an internal control gene. Primer sequences are listed below [forward primer (F), reverse primer (R)]:

NIS primers (NM000453, PrimerBank no. 4507035a1), F, 5'-TGCGGGACTTTGCAGTACATT, R, 5'-TGCAGATAATTCCGGTGGACA; hCGß primers (NM033142, PrimerBank no. 15451750a1), F, 5'-GGTGTGCAACTACCGCGAT; R, 5'-GGAGTCGGGATGGACTTGGA; ß2M primers (NM 004048, PrimerBank no. 4757826a1), F, 5'-GGCTATCCAGCGTACTCCAAA; R, 5'-CGGCAGGCATACTCATCTTTTT; pendrin primers (NM 000441, PrimerBank no. 4505697a1), F, 5'-TGTGCTAAAGACTCTTGTGCC; R, 5'-CACCACTGGAAAAGGTCCAAC.

The real-time PCR method was validated by using serially diluted cDNA as a standard curve. To quantify the gene expression profile in each sample, the efficiency of each standard curve was determined by its slope and comparative threshold according to the manufacturer’s instructions. For each sample, the amount of targeted mRNA (arbitrary units) was normalized to the housekeeping gene ß2M.

Membrane protein preparation and Western blots

Membrane proteins were extracted by ProteinExtract native membrane protein extraction kit (Merck, Kilsyth, Victoria, Australia) following the company protocol. Briefly, monolayers of BeWo cells were washed twice with washing buffer. Extraction buffer I with protease inhibitors was added to the cells with gentle agitation for 10 min at 4 C, and the supernatant was discarded. The cell layers were then covered with extraction buffer II plus protease inhibitors and gently agitated for 30 min at 4 C. The supernatant containing the membrane fraction was placed into a sample tube, and membrane proteins were concentrated by Ultrafree-MC centrifugal filter unit (Millipore, Billerica, MA). Protein concentration was measured by a BCA protein assay kit (Pierce, Rockford, IL).

For electrophoresis, 30 µg of membrane samples were prepared by addition of NuPAGE sodium dodecyl sulfate sample buffer with reducing agent (NuPAGE; Novex, Invitrogen) and incubated at 70 C for 10 min. The samples were loaded in 4–12% acrylamide gradient gel (NuPAGE; Novex, Invitrogen) and transferred to a 0.45-µm nitrocellulose membrane (Bio-Rad, Hercules, CA). The membrane were blocked with 5% low-fat dry milk in Tris-buffered saline [20 mM Tris-Cl (pH 7.4), 137 mM NaCl, 0.05% Tween 20] for 45 min at room temperature. The blots were then probed with monoclonal anti-NIS antibody diluted in 1:400 (Chemicon, Temecula, CA) in blocking buffer overnight at 4 C. After washing three times with Tris-buffered saline [20 mM Tris-Cl (pH 7.4), 137 mM NaCl, 0.05% Tween 20], the membrane was probed with horseradish peroxidase-conjugated secondary antibody 1:2000 (ImmunoPure peroxidase conjugated goat antimouse IgG; Pierce) in blocking buffer, and washed as before. The signals were visualized with Super Signal West Femto maximum sensitivity substrate (Pierce) and detected by FujiFilm luminescent image analyzer LAS-3000 (FujiFilm, Tokyo, Japan). Amount of chemiluminescence of NIS bands was measured by FujiFilm Multi Gauge software (version 3.0).

Radioiodide uptake

BeWo cells were subcultured into six-well plates when approaching confluence. 125Iodide as NaI, carrier-free in NaOH, was obtained from Amersham Biosciences (Castle Hill, New South Wales, Australia). 125I uptake assay was performed as previously described (7). Briefly, NaI-treated cells were washed with medium twice for 3 min followed by a further incubation with serum-free medium for 1 h. This allowed efflux of iodide from the cells and ensured that cells were free of added iodide before 125I uptake studies. Washed cells were then incubated in 2 ml serum-free DMEM medium with 125I (1 kBq/ml) for 1 h at 37 C, solubilized in 1 ml of 1 M NaOH, and the wells rinsed with a further 1 ml NaOH. The level of 125I uptake was quantified by measuring 125I in 2 ml solubilized cell extract in a {gamma}-counter. The uptake experiments were repeated three times from separate cell cultures, and each experiment was in triplicate.

To exclude the possibility that the reduction in 125I uptake was caused by the remaining free iodide from NaI treatment, the cells were incubated with a tracer amount of 125I and washout experiments performed mimicking as closely as possible the washing steps of the uptake experiments. The amount of 125I remaining in cells was measured by a {gamma}-counter.

Immunoassay

hCG secreted into the cell culture medium was measured by sandwich immunoassay using direct chemiluminometric technology (ADVIA Centaur Total hCG assay; Bayer, Pymble, New South Wales, Australia). Two antibodies were used in the assay: goat polyclonal anti-hCG antibody and mouse monoclonal anti-hCG antibody. Each antibody reacts with a specific hCG epitope and detects both free ß-subunit hCG and ß-subunit of intact hCG. The assay was performed according to the manufacturer’s instructions.

Statistics

Statistical analysis was performed using GraphPad Prism software (version 4.0; GraphPad Software Inc., San Diego, CA). Comparison between groups was by one-way ANOVA with Bonferroni’s post hoc test to measure differences between the means of treatment groups vs. the control group. Results are expressed as mean ± SE and P < 0.05 was considered significant.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Preincubation with iodide significantly decreased NIS mRNA expression in BeWo cells in a dose- and time-dependent manner (Fig. 1AGo). The most significant effect was at 24 h after 100 µM of NaI treatment (P < 0.001). As shown in Fig. 1CGo, iodide had no effect on pendrin mRNA expression. Membrane NIS protein, measured by Western blotting, was significantly reduced in iodide-treated BeWo cells (Fig. 2Go). A major band with molecular mass of approximately 65 kDa was detected in membrane protein extracts. Two minor bands with molecular mass of 250 and 200 kDa were also visualized.


Figure 1
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  		FIG. 1. Effects of iodide on NIS mRNA and 125Iodide uptake in BeWo cells. A, Quantitative real-time PCR shows dose- and time-dependent iodide inhibition of NIS mRNA expression. Data are presented as ratio of NIS mRNA normalized to ß2M, a house keeping gene. B, 125Iodide uptake was measured after incubation with increasing doses of NaI for 3 d. Iodide inhibits 125Iodide uptake in a dose-dependent manner. C, The level of pendrin mRNA was measured from the same cDNA preparations used to examine NIS expression. There was no significant effect of iodide on pendrin. **, P < 0.01; ***, P < 0.001 (ANOVA followed by Bonferroni’s post hoc test).

 

Figure 2
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  		FIG. 2. Effects of iodide on NIS membrane protein expression in BeWo cells by Western blot. Thirty micrograms of membrane protein from BeWo cells were loaded in each lane. Under reducing conditions, an approximately 65-kDa strong band and two weaker bands of 250 and 200 kDa were detected using monoclonal anti-NIS antibody in both control and iodide-treated BeWo cells. Incubation with 100 µM of NaI for 3 d significantly reduced NIS protein expression. Densitometry data plotted in the graph were from four individual cultures, each repeated at least twice (n = 9). **, P < 0.01 (t test).

 
Functional iodide uptake was tested using 125I. We confirmed 125I uptake in BeWo cells with a inhibitory constant of 6.0 ± 0.12 µmol as reported previously (7). As shown in Fig. 1BGo, a dose-dependent inhibition of 125I uptake was seen in NaI-treated cells. To exclude the possibility that the reduction in 125I uptake was caused by residual unlabeled free iodide in the cells, washout experiments were done as described in Materials and Methods. Culture medium containing 69,200 cpm of I125 was added to each well. Only 11.67 ± 0.66 cpm (0.02%) remained after washing.

hCG mRNA was also decreased by iodide in a dose- and time-dependent manner in BeWo cells (Fig. 3AGo). Significant inhibition was observed after 24 h of treatment with NaI concentrations as low as 1 µM. Levels of hCG protein expression were also reduced in a time- and dose-dependent manner after preincubation with NaI (Fig. 3BGo).


Figure 3
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  		FIG. 3. Iodide inhibits ßhCG mRNA and protein expression in BeWo cells. A, Level of ßhCG mRNA was quantified by real-time PCR and presented as ratio of hCG mRNA normalized to ß2M. hCG mRNA expression was inhibited in a time- and dose-dependent manner by iodide. B, BeWo cells were exposed to different concentrations of NaI for 3 and 5 d. ßhCG in culture medium was measured by immunoassay. hCG protein secretion was decreased by iodide in a dose- and time-dependent manner. ***, P < 0.001 (ANOVA followed by Bonferroni’s post hoc test).

 
To examine the mechanism of iodide inhibition effects further, cells were incubated with hCG, PTU, or DTT plus iodide. hCG increased NIS expression in a dose-dependent manner. This effect was significantly reduced by coincubation with 100 µM iodide (Fig. 4Go). Coincubation with 1 mM PTU increased basal NIS mRNA expression and blocked iodide-induced inhibition of NIS mRNA expression (Fig. 5AGo) and iodide uptake (Fig. 5BGo). Similarly, PTU prevented iodide-induced inhibition of hCG expression (Fig. 5CGo). DTT also reversed iodide-mediated inhibition of NIS mRNA expression (Fig, 6Go).


Figure 4
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  		FIG. 4. Effects of iodide on hCG-regulated NIS gene expression in BeWo cells. Cells were incubated with increasing concentrations of hCG with or without 100 µM of NaI for 24 h. NIS mRNA was quantitated by real-time PCR. NIS mRNA expression was increased by hCG in a dose-dependent manner. Iodide significantly blocked hCG-stimulated NIS expression. ***, P < 0.001 (ANOVA followed by Bonferroni’s post hoc test).

 

Figure 5
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  		FIG. 5. Effects of PTU on iodide inhibition of NIS and hCG gene expression and 125Iodide uptake. A, BeWo cells were cultured with increasing concentrations of NaI ± 1000 µM of PTU for 24 h. NIS and ß2M mRNA were measured by quantitative real-time PCR. Data are presented as the percentage of NIS/ß2M mRNA relative to control. PTU increased NIS mRNA expression and blocked iodide effects on NIS mRNA expression. B, BeWo cells were cultured with NaI ± PTU and PTU alone for 3 d. NIS protein activity was measured by 125Iodide uptake. Incubation with PTU alone increased 125Iodide uptake and blocked inhibitory effects of iodide on radioiodine uptake. C, PTU also increased hCG mRNA expression and blocked iodide inhibition of hCG mRNA expression. **, P < 0.01, ***, P < 0.001 (ANOVA followed by Bonferroni’s post hoc test).

 

Figure 6
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  		FIG. 6. Effect of DTT on iodide inhibition of NIS gene expression in BeWo cells. Cells were incubated with 0.1 and 1.0 µM of DTT with or without 100 µM NaI for 24 h. NIS mRNA was measured by quantitative real-time PCR. Data are expressed as percentage of NIS/ß2M mRNA relative to controls. DTT in doses of 0.1 and 1.0 µM blocked iodide inhibition of NIS mRNA expression. The increase in NIS mRNA expression resulting from incubation with DTT alone was not statistically significant. **, P < 0.01, ***, P < 0.001 (ANOVA followed by Bonferroni’s post hoc test).

 
Viability of cells, as judged by Trypan blue exclusion and LDH release assay, was similar in all experiments (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Inhibitory effects of iodide on NIS expression and 125I uptake in BeWo cells

We previously reported significant iodide uptake by BeWo cells, with kinetic and inhibitory characteristics consistent with uptake by NIS (7). In this in vitro study, we examined the effect of iodide on NIS mRNA using polarized BeWo cells as a trophoblast model. We confirmed our previous finding of iodide uptake by BeWo cells and, importantly, demonstrated that iodide inhibits NIS mRNA expression in a dose- and time-dependent manner (Fig. 1AGo). Iodide-induced inhibition of NIS mRNA expression was associated with decreased NIS protein expression in cell membranes (Fig. 2Go) and a reduction in 125I uptake (Fig. 1BGo). These latter effects were not apparent until 3 d of incubation with iodide. This delay may relate to the half-life of NIS protein, which may be up to 4 d (15). Iodide had, however, no effect on the expression of pendrin (Fig. 1CGo), which is responsible for iodide release from these cells (7, 16). Washout experiments excluded reduction of 125I uptake by residual amounts of free iodide from iodide treatment. This indicates that the reduced 125I uptake by iodide represents effects on NIS function only.

There is only limited information about plasma inorganic iodide levels in pregnant women and the fetus. It has been reported that plasma inorganic iodide levels in pregnant women are around 0.15–0.4 µM and fetal levels around 0.3 µM (17, 18). The concentrations of sodium iodide used in this study ranged from 1 to 100 µM. We have shown that this has no effect on cell viability or pendrin gene expression. A previously published in vitro study of iodide-induced apoptosis used 10–50 mM of iodide (19), which is at least 100 times higher than that used in this study. We consider that the iodide effects are not due to iodide toxicity.

Fetal thyroid hormone synthesis depends on maternal iodide supply. This involves two steps of active iodide transport: first, placental transport of maternal iodide by trophoblast to the fetal circulation and second, transport of iodide from the fetal circulation into fetal thyroid cells for production of thyroid hormone. This active iodide transport in trophoblast and thyroid cells relies on NIS expression (7, 20).

Schroder-van der Elst et al. (14) reported up-regulation of placental NIS mRNA in iodine-deficient rats. This and our findings are consistent with a role for iodide in placental NIS gene regulation. Interestingly, cord blood thyroid hormone levels in neonates of mothers with moderate iodine deficiency and hypothyroxinemia are significantly higher than maternal levels (21, 22). Whereas no measurements of serum inorganic iodine concentrations were made in these cases, we speculate that up-regulated placental NIS expression and increased maternofetal placental iodide transport may allow the fetus to maintain normal thyroid hormone levels in the face of moderate maternal iodide deficiency. On the other hand, excessive maternal iodide concentrations may down-regulate NIS expression in placenta and reduce iodide transport to the fetus.

Inhibitory effects of iodide on hCG expression and hCG action on NIS in BeWo cells

Trophoblasts not only express NIS but also produce hCG, which shares an identical {alpha}-chain and a highly homologous ß-chain with TSH. TSH and hCG stimulate NIS expression and iodide uptake in thyroid cells (8, 9, 23), and hCG increases NIS gene and protein expression and iodide uptake in the JAr human choriocarcinoma cell line (10). We therefore examined hCG expression in cells treated with NaI. Iodide-induced inhibition of NIS expression was accompanied by inhibition of hCG mRNA and protein expression (Fig. 3Go, A and B). We did note, however, that an effect of iodide (100 µM) on NIS mRNA expression was detectable by 4 h (Fig. 1AGo), whereas iodide effects on hCG protein expression were not evident until 5 d of iodide incubation (Fig. 3AGo). It is therefore unlikely that in this study iodide inhibits NIS expression through inhibition of hCG expression. However, slow inhibition of hCG expression by iodide could subsequently reduce NIS expression.

In thyroid, TSH modulation of NIS is effected through a NIS promoter and NIS upstream enhancer through a cAMP signal transduction pathway (24). The paired-box gene 8 (PAX8) is required for activation of both the NIS promoter and NIS upstream enhancer in thyroid (25, 26). A recent study showed that PAX8 is present in placenta and BeWo cells and is stimulated by hCG through a cAMP-dependent pathway (27). It is unclear whether hCG stimulates NIS in placenta through a similar pathway to that of TSH in thyroid and whether the effect of iodide on NIS is through inhibition of the hCG signal transduction pathways. To investigate the mechanism of iodide inhibition further, we incubated cells with hCG with and without NaI. As shown in Fig. 4Go, hCG stimulated NIS expression as previously reported. This stimulation of NIS mRNA expression by hCG was inhibited by iodide (Fig. 4Go). This suggests that in this study iodide inhibition of NIS expression may be through inhibition of hCG action on NIS.

Effects of PTU and DTT on iodide inhibition of NIS in BeWo cells

Iodide inhibition of thyroidal NIS mRNA expression has previously been reported in animal models (11, 13) and a rat thyroid cell line, FRTL-5 (12). The mechanism of iodide-induced inhibition of gene expression is unknown, but it has been postulated that intracellular generation of iodine, perhaps by formation of iodocompounds (28), mediates gene modulation. PTU may inhibit formation of thyroid hormone by acting as an alternative substrate for an iodinating intermediate generated by thyroperoxidase and hydrogen peroxide (29). To examine the effects of PTU on iodide effects in placental cells, BeWo cells were exposed to PTU alone or in combination with a range of concentrations of NaI. As shown in Figs. 4Go and 5Go, PTU blocked the iodide-induced decrease in NIS and hCG mRNA expression and 125I uptake in BeWo cells. This effect of PTU may be through a mechanism similar to that in thyroid. Our finding that PTU in the absence of iodide increased NIS and hCG mRNA and iodide uptake is interesting. PTU has previously been reported to have effects on gene expression including reduction of NIS mRNA and iodide uptake in FRTL-5 cells (30), increased TPO mRNA in cultured porcine follicles (31), and thyroglobulin mRNA expression (32) in FRTL-5 cells. We acknowledge, however, that the mechanism of this effect of PTU on gene regulation is uncertain.

Accumulating evidence suggests that reduction/oxidation (Redox) balance plays an important role in cellular signaling transduction pathway by regulating DNA-binding activity of transcription factors. This balance of the Redox state is partly maintained by reactive oxygen species, e.g. hydrogen peroxide (H2O2), superoxide anion (O2–), hydroxyl radical (OH). It has been reported that the Redox state regulates PAX8 DNA-binding activity in rat thyroid FRTL-5 (33, 34). Cotransfection of HeLa cells with Redox factor-1, a nuclear enzyme mediating reduction of transcription factors, and PAX8 significantly increased the activity of NIS promoter (35). Early studies indicated that iodide inhibits H2O2 generation (36, 37). A later study suggested, however, that high concentration of iodide (>10–4 M) and longer incubation are needed for inhibition of H2O2 generation, whereas low concentration of iodide (<10–4 M) and shorter incubation times stimulate H2O2 generation in several species of thyroid slices (38). Thus, iodide may regulate the Redox state and inhibit NIS expression by altering the level of intracellular H2O2.

DTT, a potent thiol-reducing agent, had similar effects on iodide-induced inhibition of NIS expression as PTU (Fig. 6Go), although the increase in NIS expression in the absence of iodide failed to reach statistical significance (Fig. 6Go). Recent studies suggest that H2O2 interacts with cellular thiol residues in cell signaling (39, 40). The PAX8 DNA-binding activity was abolished by thiol oxidant diamide and restored by addition of DTT and thioredoxin, a cellular reducing catalyst (34). These findings suggest that the reduced form of PAX8 is functionally important for its DNA binding. We propose that iodide regulation of NIS may be through altering Redox state, but this deserves further investigation.

In summary, our in vitro study demonstrates that iodide suppresses NIS and hCG expression and inhibits hCG stimulated NIS in BeWo cells. This decreased NIS expression was restored by DTT and PTU by an as-yet-undefined mechanism. We propose that iodide regulation of NIS modulates maternofetal iodide transfer in placenta. In circumstances of iodide excess, fetal thyroid exposure would be reduced, whereas in iodine deficiency, up-regulation of iodide transfer would optimize fetal thyroid hormone production. This regulation of NIS by iodide in placenta may be through the hCG signal pathway.


   Acknowledgments
 
We thank Professor S. Manley for his useful advice and the Department of Chemical Pathology staff for performing hCG measurements and LDH assays.


   Footnotes
 
This work was supported by the Division of Chemical Pathology, Queensland Health Pathology Services, and the Royal Brisbane and Women’s Hospital Research Foundation.

Disclosure Statement: The authors have no conflicting interests.

First Published Online August 28, 2007

Abbreviations: DTT, Dithiothreitol; F, forward primer; hCG, human choriogonadotropin; LDH, lactate dehydrogenase; ß2M, ß2-microglobulin; NIS, sodium iodide symporter; PAX8, paired-box gene 8; PTU, propylthiouracil; R, reverse primer; Redox, reduction/oxidation.

Received October 30, 2006.

Accepted July 19, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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