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Endocrinology Vol. 144, No. 3 777-784
Copyright © 2003 by The Endocrine Society

ARTICLE

Regulation of Iodothyronine Deiodinases in the Pax8-/- Mouse Model of Congenital Hypothyroidism

Sönke Friedrichsen, Stephanie Christ, Heike Heuer, Martin K. H. Schäfer, Ahmed Mansouri, Karl Bauer and Theo J. Visser

Max Planck Institut für Experimentelle Endokrinologie (S.F., S.C., K.B.), D-30625 Hannover, Germany; Department of Pathology, Columbia University (H.H.), New York, New York 10032; Institut für Anatomie und Zellbiologie, Philipps Universität Marburg (M.K.H.S.), D-35407 Marburg, Germany; Max Planck Institut für Biophysikalische Chemie (A.M.), D-37077 Gottingen, Germany; and Department of Internal Medicine, Erasmus University Medical Center (T.J.V.), NL-3000 DR Rotterdam, The Netherlands

Address all correspondence and requests for reprints to: Theo J. Visser, Ph.D., Department of Internal Medicine, Room Ee 502, Erasmus University Medical Center, Dr Molewaterplein 50, 3015 GE Rotterdam, The Netherlands. E-mail: visser{at}inw3.azr.nl.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Thyroid hormones are essential for a variety of developmental and metabolic processes. Congenital hypothyroidism (CHT) results in severe defects in the development of different tissues, in particular brain. As an animal model for CHT, we studied Pax8-/- mice, which are born without a thyroid gland. We determined the expression of iodothyronine deiodinase D1 in liver and kidney, D2 in brain and pituitary, and D3 in brain, as well as serum T4, T3, and rT3 levels in Pax8-/- vs. control mice during the first 3 wk of life. In control mice, serum T4 and T3 were undetectable on the day of birth (d 0) and increased to maximum levels on d 15. In Pax8-/- mice, serum T4 and T3 remained below detection limits. Serum rT3 was high on d 0 in both groups and rapidly decreased in Pax8-/-, but not in control mice. Hepatic and renal D1 activities and mRNA levels were low on d 0 and increased in control mice roughly parallel to serum T4 and T3 levels. In Pax8-/- mice, tissue D1 activities and mRNA levels remained low. Cerebral D2 activities were low on d 0 and increased to maximum levels on d 15, which were approximately 10-fold higher in Pax8-/- than in control mice. D2 mRNA levels were higher in Pax8-/- than in control mice only on d 21. Cerebral D3 activities and mRNA levels were high on d 0 and showed a moderate decrease between d 3 and 15, with values slightly lower in Pax8-/- than in control mice. One day after the injection of 200 ng T4 or 20 ng T3/g body weight, tissue deiodinase activities and mRNA levels were at least partially restored toward control levels, with the exception of cerebral D3 activity. In conclusion, these findings show dramatic age and thyroid state-dependent changes in the expression of deiodinases in central and peripheral tissues of mice during the first 3 wk of life.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROID HORMONE is essential for the control of a number of metabolic and developmental processes (1, 2). This is especially evident for the development of the central nervous system, which is strongly impaired by thyroid hormone deficiency during the fetal and neonatal periods (2). This may even result in cretinism, a syndrome characterized by marked neurological deficits (2). The development of the brain probably requires stage-dependent and region-specific regulation of the intracellular concentrations of T3, the main receptor-active form of the hormone. The mechanisms regulating intracellular T3 levels include the supply of circulating T4 and T3, the conversion of T4 by outer ring deiodination (ORD) to T3, and the degradation of T4 and T3 by inner ring deiodination (IRD) to rT3 and 3,3'-diiodothyronine (3,3'-T2), respectively (3, 4).

Three iodothyronine deiodinases, i.e. D1, D2, and D3, have been identified. D1 is located predominantly in liver, kidney, and thyroid and is capable of catalyzing both ORD and IRD of different iodothyronines, with rT3 as the preferred substrate (3, 4). ORD of T4 by D1 in liver and kidney is thought to be a major source of plasma T3. D2 is expressed in brain, pituitary, brown adipose tissue, and (in humans) thyroid and skeletal muscle (3, 4, 5). It only catalyzes ORD with T4 as the preferred substrate and is thus responsible for local T3 production in some of these tissues (brain and pituitary), whereas in other tissues (thyroid and muscle) it may contribute to the production of plasma T3. D3 is located primarily in brain, skin, fetal tissues, placenta, and the pregnant uterus (3, 4, 6, 7); it catalyzes the IRD and, thus, the inactivation of both T4 and T3. In the fetus, it seems to protect growing tissues against exposure to undue T3 levels.

All deiodinases have been characterized as homologous approximately 30-kDa selenoproteins (3, 4). Besides the effects of selenium intake (8), these enzymes are also regulated by thyroid state, as indicated by studies using surgically or chemically manipulated animals (3, 4, 9, 10, 11, 12, 13). As an alternative approach we decided to study the Pax8-/- mouse, which represents an elegant animal model for congenital hypothyroidism (CHT). These mice, recently generated by Mansouri et al. (14), are devoid of the paired box-transcription factor Pax8, which is required for the development of follicular structures in the thyroid (15, 16). Thus, Pax8-/- mice are unable to produce thyroid hormones. Compared with other models of experimentally induced hypothyroidism these athyroid animals offer the advantage that the effect of fetal and neonatal hypothyroidism on the expression of tissue deiodinases can be studied in the presence of maternal euthyroidism. Pax8-/- mice have an apparently normal phenotype at birth, but show strongly retarded growth and do not survive beyond weaning (14). No defects have been observed in other structures expressing Pax8, such as the developing kidney or spinal cord (14). This might be due to the redundant function of other Pax genes.

Here we report on our findings regarding the regulation of D2 and D3 expression in brain, D2 expression in pituitary, and D1 expression in the liver and kidney of Pax8-/- vs. wild-type littermates during the first 3 wk of life.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and treatments
Animal procedures were approved by the animal welfare committee of the Medizinische Hochschule Hannover. Male and female Pax8+/- mice (14) were kept at constant temperature (22 C) and light cycle (12-h light, 12-h dark) and provided with standard laboratory chow and tap water ad libitum. Genotypes of the pups were determined by Southern blot analysis of genomic DNA isolated from small tail samples. DNA was digested with PstI, size-separated on a 1% TBE (90 mM Tris, 90 mM borate, and 1 mM EDTA, pH 8.4) agarose gel, and then capillary-transferred to a nylon membrane (Hybond N, Amersham, Freiburg, Germany). The membranes were hybridized with probes specific for the neomycin gene and the replaced exon 4 of the Pax8 gene, and then washed twice in 20 mM sodium phosphate (pH 7.2) and 1% sodium dodecyl sulfate at 60 C and once at 65 C. The signals were analyzed using a Fujix BAS 1000 phosphorimager (Fuji Photo Film Co., Ltd., Dusseldorf, Germany).

Experiments were performed with young animals kept with their mother. Pax8-/- mice were studied in comparison with their Pax8+/+ and Pax8+/- littermates. To analyze the animals at later stages, litter size was reduced to four pups to increase the number of surviving Pax8-/- mice. If indicated, Pax8-/- animals received a single sc injection of T4 (200 ng/g body weight) or T3 (20 ng/g body weight) on d 20, and they were killed on d 21. These doses are 10-fold higher than the daily dose required for long-term replacement of hypothyroid rats (11). Alternatively, Pax8-/- mice received daily sc injections of 20 ng T4/g body weight from postnatal d 3 onward. Animals were killed by decapitation, and tissues were isolated quickly, frozen in liquid nitrogen, and stored at -80 C until further processing. Trunk blood was collected, and serum was obtained by centrifugation and stored at -80 C.

Northern blotting
For each experimental group, frozen brain, liver, and kidney samples from 3 animals and pituitaries from 10 animals were pooled and crushed with pestle and mortar on dry ice. Polyadenylated (poly-A) RNA was isolated from brain, kidney, and pituitary using oligo(deoxythymidine) Dynabeads (Deutsche DynAl, Hamburg, Germany) as suggested by the supplier. Total RNA was isolated from liver using peqGOLD RNApure (Peqlab, Erlangen, Germany) following the instructions of the manufacturer. RNA samples were size-fractionated on a denaturing formaldehyde-agarose gel, capillary-transferred to Hybond XL membrane (Amersham Pharmacia Biotech, Arlington Heights, IL), and analyzed under high stringency conditions. Probes were generated by PCR using cDNA from mouse brain (D2 and D3) and liver (D1), and represented nucleotides (nts) 40–389 of D1 cDNA (17) (GenBank accession no. NM007860), nts 131-1045 of D2 cDNA (18) (GenBank accession no. AF096875), and nts 43–385 of D3 (19) (GenBank accession no. AA389415). The cDNA probes were labeled with [{alpha}-32P]deoxy-CTP using random primers. Hybridization was carried out at 42 C in UltraHyb solution (Ambion, Inc., Austin, TX). After extensive washing to final stringencies of 0.2x SSPE [0.15 M NaCl, 0.01 M sodium phosphate, and 1 mM EDTA (pH 7.4)]/0.3% sodium dodecyl sulfate for 30 min at 59 C, signals were detected by exposure to x-ray film and quantified by phosphorimaging. Similarly, cyclophilin mRNA was determined to confirm the integrity and uniformity of RNA loading (20).

Iodothyronine deiodinase activity assays
Tissues were homogenized in 10 vol or 1 ml (pituitary) 0.1 M phosphate (pH 7.2), 2 mM EDTA, and 1 mM dithiothreitol (P100E2D1 buffer). Aliquots of homogenates were snap-frozen and stored at -80 C until analysis of enzyme activities. Deiodinase activities were assayed by monitoring the preferred reaction catalyzed by the different isoenzymes, i.e. ORD of rT3 by D1, ORD of T4 by D2, and IRD of T3 by D3 (3, 4, 21, 22). D1 and D2 activities were assayed by measurement of the release of radioiodide from outer ring-labeled substrates, and D3 activity was assayed by HPLC analysis of the formation of radioactive 3,3'-T2 and 3'-iodothyronine (3'-T1) from outer ring-labeled T3 (21, 22).

D1 activity assay.
Liver or kidney homogenates (0.1 mg protein/ml) were incubated for 30 min at 37 C with 0.1 µM rT3 and 105 cpm [3',5'-125I]rT3 in 0.1 ml P100E2D10 buffer. Blank incubations were carried out in the absence of homogenate. The reactions were stopped by adding 0.1 ml ice-cold 5% BSA in water, and radioactive iodothyronines were precipitated by adding 0.5 ml ice-cold 10% (wt/vol) trichloroacetic acid in water. After centrifugation, radioiodide was further isolated from the supernatants on Sephadex LH-20 minicolumns as previously described (22). The deiodinase activity of homogenates was corrected for nonenzymatic deiodination observed in the blanks.

D2 activity assay.
Homogenates of cerebrum (~1 mg protein/ml), cerebellum (~1 mg protein/ml), or pituitary (~25 µg protein/ml) were incubated for 60 min at 37 C with 1 nM (105 cpm) [3',5'-125I]T4 in the presence of 100 nM T3 to block D3, in the presence of 0.1 mM propylthiouracil to block any D1 activity, and in the absence or presence of 100 nM unlabeled T4 to saturate D2 in 0.1 ml P100E2D25 buffer. Blank incubations were carried out in the absence of homogenate. Release of 125I- was determined and corrected for nonenzymatic deiodination as described above. The presence of 100 nM unlabeled T4 almost completely blocked radioiodide release, indicating that this indeed represented low Km D2 activity.

D3 activity assay.
Homogenates of cerebrum or cerebellum (~1 mg protein/ml) were incubated for 60 min at 37 C with 1 nM (2 x 105 cpm) [3'-125I]T3 in the absence or presence of 100 nM unlabeled T3 to saturate D3 in 0.1 ml P100D2D50 buffer. Blank incubations were carried out in the absence of homogenate. The reactions were stopped by adding 0.1 ml ice-cold methanol on ice. The mixtures were centrifuged, and 0.1 ml of the supernatants were mixed with 0.1 ml 0.02 M ammonium acetate (pH 4) and analyzed by HPLC as previously described (22). Samples of 0.1 ml were applied to a 250 x 4.6-mm Symmetry C18 column (Waters Corp., Etten-Leur, The Netherlands) connected to an Alliance HPLC system (Waters Corp.) and eluted isocratically with a mixture of acetonitrile and 0.02 M ammonium acetate (33:67, vol/vol) at a flow rate of 1.2 ml/min. Radioactivity in the eluate was monitored on-line using a Radiomatic A-500 flow scintillation detector (Packard, Meriden, CT). Conversion of labeled T3 to radioactive 3,3'-T2 and, eventually, 3'-T1 was corrected for nonenzymatic deiodination as observed in the blanks. In the presence of 100 nM unlabeled T3, the IRD of radioactive T3 was almost completely inhibited, indicating that this represented low Km D3 activity.

Hormone measurements
Serum T4, T3, and rT3 were determined by RIA. 125I-Labeled iodothyronines were obtained from Amersham Pharmacia Biotech. T4 antiserum was obtained from Sigma-Aldrich (St. Louis, MO), and T3 and rT3 antisera were produced in the Rotterdam laboratory (23). Final antibody dilutions were 1:20,000 for T4, 1:250,000 for T3, and 1:150,000 for rT3. The sample volume was 10 µl for T4, 20 µl for T3, and 25 µl for rT3, and incubation mixtures were prepared in 0.5 ml RIA buffer (0.06 M barbital, 0.15 M HCl, 0.1% BSA, and 0.6 g/liter 8-anilino-1-naphthalenesulfonic acid; Sigma-Aldrich). Mixtures were incubated in duplicate overnight at 4 C, and antibody-bound radioactivity was precipitated using Sac-Cel cellulose-coupled second antibody (IDS, Boldon, UK). The lower limits of detection amounted to less than 2 nmol T4/liter, less than 0.08 nmol rT3/liter, and less than 0.14 nmol T3/liter. Within- and between-assay coefficients of variation were 2–8% and 5–10% for T4, 2–6% and 8% for T3, and 3–4% and 9–16% for rT3, respectively.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Growth retardation of Pax8-/- mice
Female and male Pax8+/- mice showed normal fertility and reproductive capacity. Pregnancy was uneventful, and litter size was normal. The litter consisted of Pax8+/+, Pax8+/-, and Pax8-/- pups in Mendelian ratio (data not shown), but Pax8-/- pups were overrepresented in newborns with very low body weight. Of 77 animals with a birth weight in the lower tertile, 58 were identified as Pax8-/-, whereas only 8 of 156 animals with a birth weight in the upper tertile were identified as Pax8-/-. Postnatal growth of Pax8-/- pups was variable, but clearly retarded compared with Pax8+/+ and Pax8+/- littermates. Forty to 50% of the Pax8-/- mice died within the first 3 d of life, another 25% died in the remainder of the first week, about 10% died in the second week, and approximately 20% reached the age of 21 d (Fig. 1Go). To increase the number of surviving animals at later stages, litter size was reduced to 4 pups, but even then survival beyond 21 d was exceptional. Therefore, we focused our study on the first three postnatal weeks.



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  		Figure 1. Representative growth curves for a wild-type mouse, a Pax8+/- mouse, and a Pax8-/- mouse treated by daily injections of 20 ng T4/g body weight from d 3 onward. For untreated Pax8-/- mice, three typical growth curves of individual animals are shown. For untreated Pax8-/- mice, shown are three typical growth curves of individual animals that died during the second and third weeks after birth.

 
Serum iodothyronine levels in Pax8-/- vs. control mice
The profiles of serum T4, T3, and rT3 levels during the first 3 wk of life in control mice are shown in Fig. 2Go. On the day of birth (d 0), serum T4 and T3 were at or below the limits of detection in both control and Pax8-/- animals. In the control animals, serum hormone levels increased to peak levels on d 15. Thereafter, both serum T4 and T3 declined to reach near-adult levels on d 21 (adult serum T4, 63.1 ± 13.9 nmol/liter; T3, 1.15 ± 0.25 nmol/liter; mean ± SD). In the Pax8-/- animals, serum T4 and T3 remained undetectable at all ages investigated. In contrast to T4 and T3 levels, serum rT3 was high on d 0 in both control and Pax8-/- pups (Fig. 2Go). In the control animals, serum rT3 decreased in the first postnatal week to about 30% of that on d 1, to increase subsequently in parallel with serum T4 to peak levels on d 13, but with a steeper decline thereafter. In the Pax8-/- animals, serum rT3 decreased very rapidly after birth to levels below the detection limit on d 3.



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  		Figure 2. Serum T4 (A), T3 (A), and rT3 (B) levels during postnatal development in normal mice and serum rT3 in Pax8-/- mice (B) are shown. As serum T4, T3, and rT3 were identical in Pax+/+ and Pax+/- mice, they were combined into one control group. In Pax8-/- mice, T4 and T3 levels were under the detection limit. Values represent the mean ± SD of at least five determinations.

 
Twenty-four hours after the last injection, circulating hormone levels in Pax8-/- mice treated from d 3–20 with 20 ng T4/g body weight·d were 105 ± 3 nmol T4/liter and 1.12 ± 0.05 nmol T3/liter, compared with 79.3 ± 13 nmol T4/liter and 0.98 ± 0.16 nmol T3/liter in wild-type animals. After a single injection of 200 ng T4/g body weight, values were 465 ± 142 nmol T4/liter and 2.05 ± 0.6 nmol T3/liter. The serum T3 concentration in Pax8-/- mice treated with a single dose of 20 ng T3/g body weight was 32 ± 13 nmol/liter.

Liver and kidney D1 expression
The postnatal profiles of D1 activities and mRNA levels in liver and kidney of wild-type and Pax8-/- animals are shown in Fig. 3Go. In control animals, D1 activities and mRNA levels in both liver and kidney were very low on the day of birth and increased dramatically until maximum values were reached after the second week. In Pax8-/- mice, hepatic and renal D1 activities and mRNA levels were also very low on the day of birth and remained low throughout the 3-wk period.



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  		Figure 3. D1 mRNA expression and enzyme activity in liver and kidney of wild-type and Pax8-/- mice during postnatal development and after treatment of 20-d-old Pax8-/- mice for 24 h with T4 or T3. Kidneys (A and C) and livers (B and D) were collected from wild-type and Pax8-/- at the postnatal stages indicated and from 21-d-old Pax8-/- mice treated on d 20 with 200 ng T4/g body weight [21d T4 (20 )] or 20 ng T3/g body weight [21d T3 (20 )]. Poly-A-enriched RNA from kidney (A) and total RNA from liver (B) were prepared and subjected to Northern blot analysis as described in Materials and Methods. After hybridization with a radioactively labeled D1 cDNA fragment, the membranes were exposed to x-ray films. Equivalence of loading and transfer to membranes were monitored by hybridization with a cyclophilin probe (lower panels). Aliquots from the same tissue collection were used to determine D1 activity in kidney (C) and liver (D) homogenates. Values represent the mean ± SD of determinations on three independent tissue preparations.

 
After administration of a single injection of T4 (200 ng/g body weight) or T3 (20 ng/g body weight) to 20-d-old Pax8-/- mice, D1 mRNA levels in both liver and kidney increased within 24 h to or above values in control animals, whereas D1 activities increased to about half of those observed in control animals. Daily injections of Pax8-/- mice with a more physiological dose of T4 (20 ng/g body weight) from d 3–20 led to normalization of mRNA levels as well as an increase in D1 activities to 70% of control values on d 21 (mean D1 in control, untreated Pax8-/-, and T4-treated Pax8-/- mice, 33.0, 0.2, and 22.4 pmol/min·mg protein in liver, and 19.5, 0.4, and 11.9 pmol/min·mg protein in kidney, respectively).

Brain D2 and D3 expression
Figure 4Go shows the profiles for cerebral D2 mRNA levels and enzyme activities in wild-type and Pax8-/- animals during the first 3 wk of life. In all animals, cerebral D2 activities and mRNA levels were very low on d 0. Both groups of animals showed an increase in brain D2 mRNA levels, with maximum levels obtained in control animals by d 9 and in Pax8-/- animals on d 21. Only at the latest time point were D2 mRNA levels clearly higher in Pax8-/- than control animals. Cerebral D2 activities were very low on d 0 in both groups of mice, although they were somewhat higher in Pax8-/- than control animals. Pax8-/- mice showed a dramatic increase in cerebral D2 activities until maximum levels were obtained on d 15. Brain D2 activities in control animals also showed a postnatal increase to a maximum on d 15, but they remained less than 1/10th of those in Pax8-/- mice.



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  		Figure 4. D2 and D3 mRNA expression and enzyme activity in brain of wild-type and Pax8-/- mice during postnatal development and after treatment of 20-d-old Pax8-/- mice for 24 h with T4 or T3. Brains were collected from wild-type and Pax8-/- at the postnatal stages indicated and from 21-d-old Pax8-/- mice receiving on d 20 a single injection of 200 ng T4/g body weight [21d T4 (20 )] or 20 ng T3/g body weight [21d T3 (20 )]. Poly-A-enriched RNA was prepared, size-fractionated, and blotted onto nylon membranes as described in Materials and Methods. The membranes were hybridized with radioactively labeled cDNA fragments of D2 (A) and D3 (B) and exposed to x-ray films. Equivalence of loading and transfer was monitored by hybridization with a cyclophilin probe. D2 activity (C) and D3 activity (D) were determined in homogenates prepared from aliquots of the tissue samples used for Northern blot analysis. Values represent the mean ± SD of determinations on three independent tissue preparations. Statistical analysis of differences between wild-type and Pax8-/- mice was performed by t test (two tailed). C: *, P = 0.07; **, P < 0.001; D: *, P < 0.05; **, P < 0.005.

 
A single injection of T4 (200 ng/g body weight) or T3 (20 ng/g body weight) to 20-d-old Pax8-/- mice led to a down-regulation of cerebral D2 mRNA within 24 h to lower levels than those observed in untreated control mice. The administration of T3 resulted in a modest decrease in brain D2 activity, whereas D2 activity was almost completely abolished by the T4 injection.

Figure 4Go also shows the neonatal development of cerebral D3 expression in Pax8-/- vs. wild-type animals. In both groups of mice, D3 activities as well as mRNA levels were equally high on d 0 and 3. From d 9–21, modest decreases in D3 activities and mRNA levels were observed, which were more pronounced in Pax8-/- than in control animals.

Treatment of Pax8-/- mice on d 20 with a single injection of T4 (200 ng/g body weight) or T3 (20 ng/g body weight) resulted on d 21 in an increase in cerebral D3 mRNA expression to levels similar to those in untreated control animals. D3 activity was not affected 24 h after injection of these moderate T4 and T3 doses (Fig. 4Go), although we observed full restoration of D3 activity 24 h after a single injection of a 10-fold higher dose of T4 (2 µg/ g body weight) or T3 (0.2 µg/g body weight; data not shown).

Results similar to those described for cerebral D2 and D3 expression were observed with regard to D2 and D3 mRNA expression and enzyme activity profiles in the cerebellum of Pax8-/- vs. control mice (data not shown).

D2 expression in the pituitary
D2 is also important for local T4 to T3 conversion in the pituitary and may thus be involved in the feedback control of TSH secretion as well as the regulation of other pituitary hormones (3, 4, 24, 25). We therefore determined hypophyseal D2 activities and mRNA levels in control and Pax8-/- mice on d 21 (Fig. 5Go). We observed a dramatic increase in both D2 mRNA expression and enzyme activities in pituitaries from Pax8-/- mice compared with their wild-type littermates. D1 mRNA could not be detected in pituitaries of control and Pax8-/- animals.



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  		Figure 5. D2 mRNA expression and enzyme activity in the pituitary of Pax8-/- and wild-type mice. As described in Materials and Methods, poly-A-enriched RNA prepared from pituitaries of 21-d-old wild-type and Pax8-/- mice was subjected to Northern blot analysis using a radioactively labeled D2 cDNA fragment (A) for hybridization. As a control, the membrane was also hybridized with a cyclophilin probe (lower panel). D2 enzyme activity in 21-d-old pituitaries of wild-type and Pax8-/- mice (B) was determined as described in Materials and Methods. Values represent the mean ± SD of determinations on three independent tissue preparations.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In humans, 20% of CHT is caused by thyroid hormone synthesis defects, and 80% is caused by thyroid development defects (dysgenesis). In about 20% of subjects with CHT the thyroid gland is completely absent (agenesis) (26, 27). Thyroid dysgenesis and agenesis are caused by mutations in various transcription factors, among others in Pax8 (28). We have used Pax-/- mice as a model to study the effects of CHT where the development of an athyroid fetus takes place in a euthyroid mother and within a predominantly euthyroid litter. The Pax8-/- mouse has obvious advantages over other animal models for CHT involving surgical and/or medical treatments (9, 10, 11, 12, 13). However, the hypothyroidism of the untreated Pax8-/- pup is so severe that they fail to thrive and usually die before they are 3 wk old. The severe growth impairment probably results from reduced food intake and/or reduced food utilization. This could suggest that the changes we observed in Pax8-/- vs. control mice regarding regulation of deiodinase expression may be caused only indirectly by thyroid hormone deficiency. It is known that profound alterations take place in peripheral thyroid hormone metabolism in nonthyroidal illness and fasting (3, 4, 29), conditions that may well apply to the neonatal Pax8-/- mouse. However, we also show that most changes in deiodinase expression in the neonatal Pax8-/- are acutely reversed by the administration of T4 or T3. Therefore, the effects of homozygous Pax8 gene deletion we observed appear mostly as a direct result of the absence of thyroid hormone rather than indirect effects mediated by illness or starvation. It should also be emphasized that Pax8-/- mice develop without overt defects if adequate thyroid hormone replacement therapy is started early after birth (13). This also results in normalization of the expression of the deiodinases in the different tissues.

Not only is postnatal growth impaired in Pax8-/- mice, but their birth weight is also lower than that of control littermates, suggesting that prenatal thyroid hormone levels are lower in Pax8-/- mice than in control animals. This seems to be contradicted by our findings that serum T4 and T3 levels even in normal mice are virtually undetectable on the day of birth, although this does not exclude that significant thyroid hormone levels may be present in fetal tissues of control mice. Indeed, D1 activity and mRNA levels in liver and kidney immediately after birth appear higher in control than in Pax8-/- pups, suggesting that liver and kidney are exposed prenatally to somewhat higher thyroid hormone levels in control than in Pax8-/- fetuses. The extremely low serum T4 and T3 levels on the day of birth in both control and Pax8-/- mice suggests minor placental transfer of these hormones at least in the later stages of gestation in the mouse. This is in contrast to the prominent maternal-fetal transfer of thyroid hormone through the human placenta, as documented in CHT infants with thyroid agenesis (30). The lack of a postnatal increase in serum T4 and T3 in the Pax8-/- mice further suggests negligible transfer of thyroid hormone from mother to pup via the milk, which has recently been demonstrated in humans (31).

In contrast to the extremely low T4 and T3 levels immediately postpartum, serum rT3 was high in both control and Pax8-/- pups on d 0. This indicates that fetal serum rT3 is not derived from secretion of rT3 by the fetal thyroid or from IRD of T4 secreted by the fetal thyroid, but from IRD of maternal T4 in the placenta and perhaps also the uterus (3, 4, 7, 32). Due to the disruption of the maternal supply of T4 and placental metabolites, serum rT3 decreases in the Pax8-/- neonate. The rapidity of this decrease is remarkable, as the main pathway of rT3 clearance (D1-mediated deiodination) is lacking, whereas D2 activity is still very low. Furthermore, D3 does not deiodinate rT3. Therefore, rT3 must be cleared in the Pax8-/- mouse by alternative routes, such as conjugation, in particular sulfation (33). Serum rT3 disappears much slower in control pups and subsequently increases, apparently due to the increase in neonatal thyroid function, as indicated by the postnatal increase in serum T4 and T3 levels. This increase in serum rT3 probably reflects the IRD of T4 in tissues with high D3 activity, such as brain and skin (3, 4, 6, 34, 35).

Our findings in the neonatal Pax8-/- mouse are in agreement with previous studies of regulation of the expression of D1 in rodents by thyroid state (9, 10, 11, 12, 13). Firstly, the ontogenic profiles of D1 activities and mRNA levels in liver and kidneys of control mice closely follow the postnatal increases in serum T4 and T3 levels. Secondly, the lack of postnatal increase in serum T4 and T3 in Pax8-/- mice is associated with an almost complete loss of the expression of D1 activity and mRNA levels in both liver and kidney. The very low, but detectable, D1 mRNA and activity levels in liver and kidney of 9- to 15-d-old Pax8-/- mice suggest minor thyroid hormone-independent D1 expression. Thirdly, replacement of Pax8-/- mice with relatively low doses of T4 or T3 results within 24 h in an increase in D1 mRNA in both liver and kidney to levels higher than those in control animals, although D1 activities are increased to about 50% of control activities. Most likely, this is due to the delay in production of functional D1 protein relative to the thyroid hormone-induced increase in D1 mRNA, which has previously also been observed in thyroid hormone-treated hypothyroid rats (9). The correlation between hepatic and renal D1 mRNA levels and enzyme activities during postnatal development in normal mice as well as after treatment of Pax8-/- mice with T4 or T3 suggests that D1 expression is regulated by thyroid hormone predominantly at the pretranslational level. In combination with the identification of T3 receptor-binding response elements in the D1 gene promoter (36, 37), these results indicate that thyroid hormone regulation of D1 expression is largely exerted by T3 at the level of gene transcription. It is remarkable, then, that the hepatic and renal expression of D1 is under positive control of what is thought to be its physiologically most important product, T3.

We found that cerebral D2 activities are much higher in hypothyroid Pax8-/- mice than in their euthyroid littermates. This was only accompanied by increased D2 mRNA expression at the latest age tested (d 21). Before this age, thyroid state-dependent regulation of D2 expression was largely exerted at the translational or posttranslational level. These results agree with previous findings, largely in rats, indicating that the negative control of D2 expression in brain and pituitary by thyroid hormone involves two different mechanisms (3, 4, 10, 38, 39, 40, 41, 42). Firstly, down-regulation of D2 mRNA expression by thyroid hormone is probably mediated by the nuclear T3 receptor (10, 42). It is unknown whether this involves the suppression of D2 gene transcription by interaction of the T3 receptor with a negative T3 receptor-binding response element in the promoter region of his gene. Secondly, and more importantly, D2 appears to undergo substrate-induced enzyme inactivation, which is exerted largely by the substrates T4 and rT3, whereas T3 is much less active in this respect (38, 39, 40, 41, 42). In rat glial cells, down-regulation of D2 activity by T4 and rT3 is associated with the internalization of plasma membrane-bound D2 or a subunit(s) thereof (39). In pituitary cells and D2 cDNA-transfected cells, substrate-induced degradation of D2 involves the ubiquitination of the protein (40, 41). The exact molecular transformation of the D2 protein induced by substrate and leading to its degradation remains to be demonstrated.

Also in agreement with previous findings in rats, we show that cerebral D2 expression peaks in the postnatal period (6, 43, 44). Maximum D2 activity is observed on d 15, being approximately 10-fold higher in Pax8-/- than control mice. However, by the day of birth, already there appears to be a significant difference in the (low) D2 activities between Pax8-/- and control mice. It remains unknown whether this is due to small differences in brain iodothyronine levels between Pax8-/- and control pups in the perinatal period. At birth, rT3 appears to be the major serum iodothyronine, but our data do not allow a conclusion regarding possible differences in serum rT3 levels between Pax8-/- and control newborns, which may be associated with differences in D2 protein inactivation rates.

In the pituitary on d 21 we also observed much higher D2 activities in Pax8-/- than control mice. The exact physiological role for D2 expression in the pituitary is still unsettled. Physiological studies have made a case for the importance of local T4 to T3 conversion in the pituitary for the negative feedback regulation of TSH secretion by thyroid hormone (3, 4, 24, 25). However, negative regulation of D2 activity by thyroid hormone would dampen changes in intracellular T3 levels in the thyrotrope with changes in thyroid activity and thus interfere with a proper feedback control of TSH secretion. D2 is also expressed in other pituitary cells, such as somatotropes, lactotropes, and gonadotropes, the functions of which may also depend on local T4 to T3 conversion (25, 45). However, it is worthwhile mentioning in this respect that pituitaries of Pax-/- mice show a dramatic reduction in the number of GH, PRL, and gonadotropin-producing cells and a dramatic increase in the number of TSH-producing cells (our unpublished observations). These findings suggest that in the pituitary of Pax8-/- mice, D2 is predominantly located in the thyrotrope. Colocalization studies of pituitary hormones and D2 should provide an answer to this important question.

Finally, our findings are in agreement with previous reports on the ontogeny of brain D3 activity in rodent brain, which have indicated that cerebral D3 activities are highest in the fetal period (6, 35, 43). In our neonatal mice, high D3 activities and mRNA levels were observed in the first 3 d of life, followed by a modest decrease until d 15 to remain at that level until d 21. On d 0–3, no difference was seen in D3 expression between Pax8-/- and control pups, but D3 activity and mRNA levels were lower on d 15 and 21 in Pax8-/- mice than in control animals. Also in cerebellum we found significantly lower D3 expression in the Pax8-/- neonates than in control mice. Therefore, these findings are in line with other studies showing positive control of D3 expression in brain by thyroid state (13, 46). In the ontogenic profiles, brain D3 activities and mRNA levels appear to run in parallel in both groups of animals, suggesting that both developmental stage- and thyroid hormone-dependent regulation of D3 expression are exerted at the pretranslational level. D3 expression is not under control of thyroid hormone in all tissues. For instance, this is not the case in placenta (47, 48). The mechanism of thyroid hormone regulation of D3 expression in tissues such as the brain remains to be determined. The physiological function of the expression of high D3 activities in placenta, pregnant uterus, and different fetal tissues is not entirely understood; it may serve to protect growing tissues against exposure to undue T3 levels until the active hormone is required for induction of tissue differentiation.

In conclusion, we have presented novel data regarding the ontogeny of the different deiodinases in tissues of normal mice as well as in congenitally hypothyroid animals. The regulation of the expression of these enzymes is in general agreement with previous findings in other experimental animal models. D1 in liver and kidney and D3 in brain are under positive control of thyroid hormone, whereas D2 expression in brain and pituitary is under negative control of thyroid state. These adaptations appear to serve the purpose of maintaining T3 levels in certain tissues in different thyroid states. Thus, in hypothyroidism, less T4 is degraded by D1 and D3 to increase its availability for local conversion to T3 by D2, the activity of which is increased, in critical tissues such as the brain.


   Acknowledgments
 
We thank Ellen Kaptein, Hans van Toor, and Melanie Kraus for expert technical assistance, and Valerie Ashe for linguistic help.


   Footnotes
 
This work was supported by the Deutsche Forschungsgemeinschaft.

Abbreviations: CHT, Congenital hypothyroidism; D1, D2, or D3, type I, II, or III iodothyronine deiodinase; IRD, inner ring deiodination; nts, nucleotides; ORD, outer ring deiodination; poly-A, polyadenylated; T1, monoiodothyronine; T2, diiodothyronine.

Received July 15, 2002.

Accepted for publication November 15, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Visser TJ 2002 Biosynthesis, transport, metabolism and action of thyroid hormones. In: Wass J, Shalet S, eds. Oxford textbook of endocrinology and diabetes. Oxford: Oxford University Press; 287–301
  2. Bernal J 2002 Action of thyroid hormone in brain. J Endocrinol Invest 25:268–288[Medline]
  3. Leonard JL, Koehrle J 2001 Intracellular pathways of iodothyronine metabolism. In: Braverman LE, Utiger RD, eds. The thyroid. Philadelphia: Lippincott-Williams & Wilkins; 136–173
  4. Bianco AC, Salvatore D, Gereben B, Berry MJ, Larsen PR 2002 Biochemistry, cellular and molecular biology, and physiological roles of the iodothyronine selenodeiodinases. Endocr Rev 23:38–89[Abstract/Free Full Text]
  5. Salvatore D, Bartha T, Harney JW, Larsen PR 1996 Molecular biological and biochemical characterization of the human type 2 selenodeiodinase. Endocrinology 137:3308–3315[Abstract]
  6. Bates JM, St. Germain DL, Galton VA 1999 Expression profiles of the three iodothyronine deiodinases, D1, D2, D3, in the developing rat. Endocrinology 140:844–851[Abstract/Free Full Text]
  7. Galton VA, Martinez E, Hernandez A, St Germain EA, Bates JM, St Germain DL 1999 Pregnant rat uterus expresses high levels of the type 3 iodothyronine deiodinase. J Clin Invest 103:979–987[Medline]
  8. Bates JM, Spate VL, Morris JS, St. Germain DL, Galton VA 2000 Effects of selenium deficiency on tissue selenium content, deiodinase activity, and thyroid hormone economy in rat during development. Endocrinology 141:2490–2500[Abstract/Free Full Text]
  9. O’Mara BA, Dittrich W, Lauterio TJ, St. Germain DL 1993 Pretranslational regulation of type I 5'-deiodinase by thyroid hormones and in fasted and diabetic rats. Endocrinology 133:1715–1723[Abstract]
  10. Burmeister LA, Pachucki J, St Germain DL 1997 Thyroid hormones inhibit type 2 iodothyronine deiodinase in the rat cerebral cortex by both pre- and posttranslational mechanisms. Endocrinology 138:5231–5237[Abstract/Free Full Text]
  11. Escobar-Morreale HF, Obregon MJ, Hernandez A, Escobar del Rey F, Morreale de Escobar G 1997 Regulation of iodothyronine deiodinase activity as studied in thyroidectomized rats infused with thyroxine or triiodothyronine. Endocrinology 138:2559–2568[Abstract/Free Full Text]
  12. Tu HM, Kim SW, Salvatore D, Bartha T, Legradi G, Larsen PR, Lechan RM 1997 Regional distribution of type 2 thyroxine deiodinase messenger ribonucleic acid in rat hypothalamus and pituitary and its regulation by thyroid hormone. Endocrinology 138:3359–3368[Abstract/Free Full Text]
  13. Tu HM, Legradi G, Bartha T, Salvatore D, Lechan RM, Larsen PR 1999 Regional expression of the type 3 iodothyronine deiodinase messenger ribonucleic acid in the rat central nervous system and its regulation by thyroid hormone. Endocrinology 140:784–790[Abstract/Free Full Text]
  14. Mansouri A, Chowdhury K, Gruss P 1998 Follicular cells of the thyroid gland require Pax8 gene function. Nat Genet 19:87–90[CrossRef][Medline]
  15. Plachow D, Chowdhury K, Walther C, Simon D, Guenet JL, Gruss P 1990 Pax8, a murine paired box gene expressed in the developing excretory system and thyroid gland. Development 110:643–651[Abstract/Free Full Text]
  16. Magliano M, Di Lauro R, Zannini M 2000 Pax8 has a key role in thyroid cell differentiation. Proc Natl Acad Sci USA 97:13144–13149[Abstract/Free Full Text]
  17. Maia AL, Berry MJ, Sabbag R, Harney JW, Larsen PR 1995 Structural and functional differences in the DIO1 gene in mice with inherited type 1 deiodinase deficiency. Mol Endocrinol 9:969–980[Abstract]
  18. Davey JC, Schneider MJ, Becker KB, Galton VA 1999 Cloning of a 5.8 kb cDNA for a mouse type 2 deiodinase. Endocrinology 140:1022–1025[Abstract/Free Full Text]
  19. Hernandez A, Lyon GJ, Schneider MJ, St. Germain DL 1999 Isolation and characterization of the mouse gene for the type 3 iodothyronine deiodinase. Endocrinology 140:124–130[Abstract/Free Full Text]
  20. Danielson PE, Forss-Petter S, Brow MA, Calavetta L, Douglass J, Milner RJ, Sutcliffe JG 1988 p1B15: A cDNA clone of the rat mRNA encoding cyclophilin. DNA 7:261–267[Medline]
  21. Koehrle J 2002 Iodothyronine deiodinases. Methods Enzymol 347:125–167[Medline]
  22. Richard K, Hume R, Kaptein E, Sanders JP, van Toor H, de Herder WW, den Hollander JC, Krenning EP, Visser TJ 1998 Ontogeny of iodothyronine deiodinases in human liver. J Clin Endocrinol Metab 83:2868–2874[Abstract/Free Full Text]
  23. Visser TJ, Docter R, Hennemann G 1977 Radioimmunoassay of reverse triiodothyronine. J Endocrinol 73:395–396[Abstract/Free Full Text]
  24. Larsen PR 1982 Thyroid-pituitary interaction: feedback regulation of thyrotropin secretion by thyroid hormones. N Engl J Med 306:23–32[Medline]
  25. Koehrle J 2000 Thyroid hormone metabolism and action in the brain and pituitary. Acta Med Austr 27:1–7[CrossRef]
  26. Krude H, Biebermann H, Schnabel D, Ambrugger P, Gruters A 2000 Molecular pathogenesis of neonatal hypothyroidism. Horm Res 53(Suppl 1):12–18
  27. Kopp P 2002 Perspective: genetic defects in the etiology of congenital hypothyroidism. Endocrinology 143:2019–2024[Abstract/Free Full Text]
  28. Macchia PE, Lapi P, Krude H, Pirro MT, Missero C, Chiovato L, Souabni A, Baserga M, Tassi V, Pinchera A, Fenzi G, Gruters A, Busslinger M, Di Lauro R 1998 PAX8 mutations associated with congenital hypothyroidism caused by thyroid dysgenesis. Nat Genet 19:83–86[CrossRef][Medline]
  29. Docter R, Krenning EP, de Jong M, Hennemann G 1993 The sick euthyroid syndrome: changes in thyroid hormone serum parameters and hormone metabolism. Clin Endocrinol (Oxf) 39:499–518[Medline]
  30. Vulsma T, Gons MH, de Vijlder JJ 1989 Maternal-fetal transfer of thyroxine in congenital hypothyroidism due to a total organification defect or thyroid agenesis. N Engl J Med 321:13–16[Abstract]
  31. van Wassenaer AG, Stulp MR, Valianpour F, Tamminga P, Ris Stalpers C, de Randamie JS, van Beusekom C, de Vijlder JJ 2002 The quantity of thyroid hormone in human milk is too low to influence plasma thyroid hormone levels in the very preterm infant. Clin Endocrinol (Oxf) 56:621–627[CrossRef][Medline]
  32. Roti E, Gnudi A, Braverman LE 1983 The placental transport, synthesis and metabolism of hormones and drugs which affect thyroid function. Endocr Rev 4:131–149[CrossRef][Medline]
  33. Visser TJ, Kaptein E, Glatt H, Bartsch I, Hagen M, Coughtrie MWH 1998 Characterization of thyroid hormone sulfotransferases. Chem Biol Interact 109:279–291[CrossRef][Medline]
  34. Huang T, Chopra IJ, Beredo A, Solomon DH, Chua Teco GN 1985 Skin is an active site of inner ring monodeiodination of thyroxine to 3,3',5'-triiodothyronine. Endocrinology 111:2106–2113
  35. Huang T, Chopra IJ, Beredo A, Solomon DH, Chua Teco GN 1988 Thyroxine inner ring monodeiodinating activity in fetal tissues of the rat. Pediatr Res 23:196–199[Medline]
  36. Toyoda N, Zavacki AM, Maia AL, Harney JW, Larsen PR 1995 A novel retinoid X receptor-independent thyroid hormone response element is present in the human type 1 deiodinase gene. Mol Cell Biol 15:5100–5112[Abstract]
  37. Jakobs TC, Schmutzler C, Meissner J, Kohrle J 1997 The promoter of the human type I 5'-deiodinase gene-mapping of the transcription start site and identification of a DR+4 thyroid hormone-responsive element. Eur J Biochem 247:288–297[Medline]
  38. Leonard JL, Silva JE, Kaplan MM, Mellen SA, Visser TJ, Larsen PR 1984 Acute posttranscriptional regulation of cerebrocortical and pituitary 5'-deiodinases by thyroid hormone. Endocrinology 114:998–1004[Abstract]
  39. Farwell AP, Safran M, Dubord S, Leonard JL 1996 Degradation and recycling of the substrate-binding subunit of type II iodothyronine 5'-deiodinase in astrocytes. J Biol Chem 271:16369–16374[Abstract/Free Full Text]
  40. Steinsapir J, Harney J, Larsen PR 1998 Type 2 iodothyronine deiodinase in rat pituitary cells is inactivated in proteasomes. J Clin Invest 102:1895–1899[Medline]
  41. Gereben B, Goncalves C, Harney JW, Larsen PR, Bianco AC 2000 Selective proteolysis of human type 2 deiodinase: a novel ubiquitin-proteasomal mediated mechanism for regulation of hormone activation. Mol Endocrinol 14:1697–1708[Abstract/Free Full Text]
  42. Halperin Y, Shapiro LE, Surks MI 1994 Down-regulation of type II L-thyroxine, 5'-monodeiodinase in cultured GC cells: different pathways of regulation by L-triiodothyronine and 3,3',5'-triiodo-L-thyronine. Endocrinology 135:1464–1469[Abstract]
  43. Kaplan MM, Yaskoski KA 1981 Maturational patterns of iodothyronine phenolic and tyrosyl ring deiodinase activities in rat cerebrum, cerebellum, and hypothalamus. J Clin Invest 67:1208–1214
  44. Obregon MJ, Ruiz de Ona C, Calvo R, Escobar del Rey F, Morreale de Escobar G 1991 Outer ring iodothyronine deiodinases and thyroid hormone economy: responses to iodine deficiency in the rat fetus and neonate. Endocrinology 129:2663–2673[Abstract]
  45. Tannahill LA, Visser TJ, McCabe CJ, Kachilele S, Boelaert K, Sheppard MC, Franklyn JA, Gittoes NJl 2002 Dysregulation of iodothyronine deiodinase enzyme expression and function in human pituitary tumors. Clin Endocrinol (Oxf) 56:735–743[CrossRef][Medline]
  46. Esfandiari A, Courtin F, Lennon AM, Gavaret JM, Pierre M 1992 Induction of type III deiodinase activity in astroglial cells by thyroid hormones. Endocrinology 131:1682–1688[Abstract]
  47. Emerson CH, Bambini G, Alex S, Castro MI, Roti E, Braverman LE 1988 The effect of thyroid dysfunction and fasting on placenta inner ring deiodinase activity in the rat. Endocrinology 122:809–816[Abstract]
  48. Koopdonk-Kool JM, de Vijlder JJ, Veenboer GJ, Ris-Stalpers C, Kok JH, Vulsma T, Boer K, Visser TJ 1996 Type II and type III deiodinase activity in human placenta as a function of gestational age. J Clin Endocrinol Metab 81:2154–2158[Abstract]



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