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Thyroglobulin Gene Mutations Producing Defective Intracellular Transport of Thyroglobulin Are Associated with Increased Thyroidal Type 2 Iodothyronine Deiodinase Activity

Department of Genetics (Y.K., Y.Miz., H.F., Y.Mu.), Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan; Department of Clinical Laboratory Medicine (A.H., T.Ie.), Dokkyo University School of Medicine, Mibu, Tochigi 321-0923, Japan; Department of Clinical Laboratory Medicine (K.T., K.S., M.M.), Gunma University Graduate School of Medicine, Maebashi 371-8511, Japan; and Departments of Endocrinology and Transplantation (Y.Miz., T.Im.), Endocrinology and Diabetology (Y.Miu., C.Y.), and Clinical Pathology (T.N.), Nagoya University Graduate School of Medicine, Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan

Address all correspondence and requests for reprints to: Yoshiharu Murata, M.D., Ph.D., Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan. E-mail: ymurata{at}riem.nagoya-u.ac.jp.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Context: Most patients with defective synthesis and/or secretion of thyroglobulin (Tg) present relatively high serum free T₃ (FT₃) concentrations with disproportionately low free T₄ (FT₄) resulting in a high FT₃/FT₄ ratio. The mechanism of this change in FT₃/FT₄ ratio remains unknown.

Objective: We hypothesize that increased type 2 iodothyronine deiodinase (D2) activity in the thyroid gland may explain the higher FT₃/FT₄ ratio that is frequently observed in patients with abnormal Tg synthesis.

Design: We recently identified a compound heterozygous patient (patient A) with a Tg G2356R mutation and one previously described (C1245R) that is known to cause a defect in intracellular transport of Tg. In the current study, after determining the abnormality caused by G2356R, we measured D2 activity as well as its mRNA level in the thyroid gland. We also measured the thyroidal D2 activity in three patients with Tg transport defect and in normal thyroid tissue.

Results: Morphological and biochemical analysis of the thyroid gland from patient A, complemented by a pulse-chase experiment, revealed that G2356R produces a defect in intracellular Tg transport. D2 activity but not type 1 deiodinase in thyroid glands of patients with abnormal Tg transport was significantly higher than in normal thyroid glands, whereas D2 mRNA level in patient A was comparable with that in normal thyroid glands. Furthermore, there was a positive correlation between D2 activity and FT₃/FT₄ ratios.

Conclusion: Increased thyroidal D2 activity in the thyroid gland is responsible for the higher FT₃/FT₄ ratios in patients with defective intracellular Tg transport.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
THYROID DYSHORMONOGENESIS caused by mutations in the thyroglobulin (Tg) gene is relatively rare, with an estimated prevalence of one in 100,000 newborns (1). Inherited in an autosomal recessive manner, the majority of patients have large goiters of elastic and soft consistency (2). Several mutations in the Tg gene have been reported in man (reviewed in Refs. 3 and 4). These include truncated Tg molecules due to premature termination of translation (5, 6), alternative splicing resulting in the deletion in a segment of the Tg molecule (7), or a simple amino acid substitution (a missense mutation) (8). In Japanese families, several different types of missense mutations have been reported (9).

We recently presented the clinical findings in a congenital goiter (patient A) caused by two missense mutations (C1245R and G2356R) in each allele of the Tg gene (10). The replacement of cysteine 1245 produces retention of the Tg molecule within the rough endoplasmic reticulum (RER) (8). However, the consequences resulting from the G2356R remained unknown. We now report immunohistochemical and electron microscopic studies on thyroid tissue obtained from patient A. In addition, we evaluated by pulse-chase experiments whether G2356R causes retention of the mutant Tg within a cell.

Although the degree of thyroid dysfunction varies considerably among patients with defective Tg synthesis, patients usually have a relatively high serum free T₃ (FT₃) concentration with disproportionately low free T₄ (FT₄) level. The maintenance of relatively high FT₃ levels prevent profound tissue hypothyroidism except in brain and pituitary, which are dependent on T₄ supply (11, 12), resulting in the neurological and intellectual defects in some cases (2). However, the mechanism by which higher FT₃ levels are maintained relative to those of FT₄ remains unknown. As in the majority of cases, patient A also had a high serum FT₃ concentration with relatively a low FT₄ level. Because type 2 iodothyronine deiodinase (D2) is expressed in the human thyroid gland and is postulated to play an important role as a source of plasma T₃ (13), we determined D2 activity in thyroid tissue obtained from patient A as well as two other patients with a homozygous C1245R mutation and one with a homozygous C1977S mutation. We found that it was increased in all four patients with Tg defect compared with normal thyroid tissues obtained from patients with thyroid tumors. The results suggest that in patients with Tg transport abnormality, increased thyroidal D2 activity is possibly responsible for the higher serum T₃ concentration relative to that of T₄.

	Subjects and Methods

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Case presentation

Patient A is a 38-year-old man who presented with sleep dyspnea due to a large goiter that was found at birth. When he was 3 yr old, methimazole was given and the prescription was continued until 8 yr of age, resulting in lethargy. At another medical center methimazole was stopped and levothyroxine (LT₄) was instituted. Thereafter, he continued to take LT₄. The dose was 100 µg/d when he was first seen by us. He had a recent gain of 20 kg since he stopped exercising and complained of dyspnea when supine due to neck compression. His sister had a goiter since birth, but the nonconsanguineous parents had no goiter or thyroid function abnormalities.

The patient’s height was 178 cm and weight 104 kg. Blood pressure was 132/92 mm Hg and the pulse 96 beats per minute. The thyroid was markedly enlarged, weighing approximately 300 g, diffuse, of soft texture, and nontender. A prominent vascular bruit was audible. There were no other physical findings except for a fine finger tremor. Blood count and electrolytes were normal, and urinalysis was positive for protein and sugar. The postprandial blood glucose was 368 mg/dl and glycosylated hemoglobin was 7.0%, indicating that the patient had diabetes mellitus. Serum total cholesterol was 152 mg/dl, triglyceride 333 mg/dl, and high-density lipoprotein cholesterol 31 mg/dl. Changes in serum TSH, FT₄, and FT₃ during LT₄ administration (100 µg/d) and after the cessation of LT₄ are shown in Table 1. Serum concentration of Tg was undetectable, and antibodies against Tg, thyroid peroxidase, and TSH receptor were all negative. The thyroidal uptake of ¹²³I was 93% at 3 h and did not decline after perchlorate administration. Dyspnea in the supine position worsened. He could not tolerate an attempt to further suppress serum TSH by increase in the LT₄ dose because of tachycardia and tremor. Therefore, a total thyroidectomy was performed.

Thyroid tissue was obtained from patient A as well as from two patients with homozygous C1245R mutation of the Tg gene and one patient homozygous for C1977S (8). Normal tissues were obtained from surgically resected glands of euthyroid patients with nonfunctioning thyroid adenomas (three cases) or carcinomas (two cases). In the experiment of endoglycosidase H (Endo H) digestion, the thyroid gland obtained from a patient with Graves’ disease was used as control. FT₄ and FT₃ were measured before surgery. The analyses of the Tg genes of patients was approved by the Ethics Committees at Nagoya University School of Medicine and Dokkyo University School of Medicine. Subjects gave written informed consent for participation in this study. Members of the family of patient A declined to participate in the study.

Sequencing and haplotype analysis of the Tg gene

Total RNA extracted from the thyroid gland of patient A was used to prepare Tg cDNA by RT-PCR and was directly sequenced. The RT-PCR conditions and the sequencing primers have been previously described (14).

For haplotype analysis, three cDNA fragments encompassing nucleotide positions from 3240–4931, from 4090–5892, and from 5442–7343 were amplified by RT-PCR. Products were ligated to pCR2.1 plasmid (Invitrogen Corp., Carlsbad, CA) and Escherichia coli (HB101 strain) were transformed by the obtained plasmids. Then, the amplified Tg cDNA fragments were sequenced.

Tissue preparations and morphological analysis

The thyroid glands obtained at the surgery were quickly frozen in liquid nitrogen and stored at –80 C until used for RNA extraction and the determination for D2 and D1 activities.

Tg immunohistochemistry was performed using HISTOFINE SAB-PO (R) kit (Nichirei Co., Tokyo, Japan) according to a protocol supplied by the manufacturer. In this kit, rabbit polyclonal antibody to human Tg (code 422691) was included. For electron microscopy, thyroid tissues were fixed in 2% glutaraldehyde, postfixed in osmium-tetrachloride, and dehydrated. Tissue blocks were embedded in epon and cut into ultrathin sections. After double staining with uranyl acetate and lead citrate, sections were examined by an electron microscope (JEM-2000X; JOEL, Akishima, Japan).

Endo H digestion

Approximately 20 mg of thyroid tissue was homogenized in 100 µl Tris buffer (10 mmol/liter, pH8.0) that contained a cocktail of protease inhibitors (Complete Protease Inhibitor Cocktail Set; Roche, Mannheim, Germany). The homogenate was centrifuged at 18,000 x g twice for 30 min each time, and the supernatant was used as a thyroid tissue extract. Tg contents in the homogenate was measured using a RIA kit (Eiken Chemical Co., Tokyo, Japan), and an aliquot of the thyroid extract containing 2 µg Tg was digested with 0.3 mU/liter Endo H (Roche) and then analyzed by 4–15% gradient SDS-PAGE as described previously (8).

Expression of human Tgs in HEK293 cells and a pulse-chase experiment

An expression plasmid containing the full-length Tg gene was constructed as follows. RNA was extracted from surgically isolated thyroid tissues using the RNeasy Mini kit (QIAGEN, Valencia, CA). Three segments of Tg cDNA, the nucleotide position at 16–3032, 2986–5717, and 5719–8323 (named as fragments A, B, and C, respectively), were amplified by RT-PCR. The PCR amplifications were performed using Expand High-Fidelity kit (Roche). The primers used were as follows: for fragment A, forward 5'-AGGAAGGGCCAGGAAAAT-3' and reverse 5'-AAATCTAGAAAAGCGGCGTCTCTGATA-3'; for fragment B, forward 5'-CTGGCGGCTCAGTCTACCTTA-3' and reverse 5'-AAATCTAGAATCTATGCTAGCTGACAGAAAGAGTGCTCCT-3'; and for fragment C, forward 5'-TCAGCTAGCAGAGATAACAGAGAGTGCATCC-3' and reverse 5'-AATTCTAGAGGAGCTCAAGGGCTGGTC-3'. For cloning purposes, the 5718th nucleotide of Tg cDNA was changed from cytosine to adenine, which does not alter the amino acid residue concerned. The amplified fragment A was cloned into pcDNA3.1/V5/His-TOPO plasmid (Eukaryotic TOPO TA Cloning Kit; Invitrogen) and then ligated to fragment B at BfrI and XbaI sites. This plasmid was ligated again to the fragment C at NheI and XbaI sites to obtain the full-length Tg cDNA.

To introduce the G2356R mutation, a guanine was replaced by an adenosine at nucleotide 7123 from the translation start site by PCR-based mutagenesis using the human wild-type Tg cDNA as a template. The mutation was confirmed by sequencing. The cDNA fragment containing the G2356R mutation was digested with NdeI and EcoRI and used to replace the corresponding fragment of wild-type human Tg-pcDNA3.1/V5-His-TOPO. The cDNA encoding C1245R mutant human Tg was also subcloned into pcDNA3.1/V5-His-TOPO (Invitrogen) (8). All plasmids were purified using the QIAGEN plasmid kit before transfection.

HEK293 cells were grown in 60-mm-diameter plastic dishes, and transfections were performed by adding 3.3 µg plasmid DNA per dish by the calcium phosphate method (15). Forty-eight hours after transfection, cells were labeled with [³⁵S]methionine (EXPRE³⁵S³⁵S 5.6 MBq/dish; specific activity, 43.5 TBq/mmol; PerkinElmer Life Sciences Inc., Boston, MA) for 3 h. Then, an excess of unlabeled methionine was added to each dish to stop further incorporation of labeled methionine into proteins, and the labeled proteins were chased for 0, 3, 6, and 24 h. After the chase, medium was collected and cells were lysed in 1 ml buffer A (16). Samples of medium and cell lysates were kept frozen at –20 C until used for immunoprecipitation.

Immunoprecipitation of Tg using 0.5 µl rabbit polyclonal antihuman Tg antibody (NeoMarkers, Fremont, CA) was performed as described previously (17) followed by 5–15% gradient SDS-PAGE. For control, 0.5 ml nonimmune rabbit serum was added to medium and cell lysate obtained from 0-h chase of wild-type Tg.

Measurement of D2 and D1 activities in the thyroid tissue

D2 activity in the microsomal fraction of thyroid tissue was measured as previously described (18). Thyroidal D1 activity was also measured with 1 µM [¹²⁵I]reverse T₃ as a substrate as described previously (19).

Northern blot analysis

Total RNA was extracted by the method of Chomczynski and Sacchi (20). To determine the expression levels of D2 and D1 mRNAs, we employed cRNA probes labeled with [-³²P]UTP. Details of this procedure were described elsewhere (18). After hybridization, autoradiography was performed by exposing the membranes to x-ray film (Kodak XAR-2; Eastman Kodak Co., Rochester, NY) for 24 h. The amounts of D2 and D1 mRNAs were measured by densitometry using NIH Image (version 1.61) and expressed in arbitrary units after correction for the amount of 28S rRNA.

Statistical analysis

Statistical differences were evaluated by the Student’s t test. Correlation between D2 activity and FT₃/FT₄ ratio was evaluated by the Pearson’s correlation coefficient test. P values < 0.05 were determined as significant difference.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Two mutations, C1245R and G2356R, found in each allele of the Tg gene in patient A

By sequence analysis of Tg cDNA, we found two nucleotide substitutions. One is a nucleotide replacement from thymine to cytosine at position 3790, resulting in the substitution of the normal cysteine with an arginine at codon 1245 (C1245R). This mutation was previously reported to be located on codon 1263 (8) because the signal peptide was formerly included in the codon numbering of the Tg molecule. The other mutation was a substitution of the normal guanine with an adenine at position of 7123. This substitution replaces the normal glycine with an arginine at codon 2356 (G2356R). We screened 103 random subjects for these two nucleic acid substitutions found in patient A and found none. Thus, the two substitutions found in patient A are not polymorphisms.

Because we could not obtain DNA samples from the parents of patient A, we performed haplotype analysis to determine whether two mutations found in patient A was located on different Tg alleles. The result indicates that this patient is compound heterozygous for the Tg gene (Fig. 1).

View larger version (7K): [in this window] [in a new window]   FIG. 1. Haplotype analysis of the Tg gene of patient A. Three overlapping cDNA fragments were amplified by RT-PCR. Primers used in PCR are indicated by arrows. After ligation into the pCR2.1 plasmid, clones were sequenced. Allele 1 contains a mutation (guanine to adenosine) at position of 7123 (in a square) that yields G2356R Tg, whereas allele 2 includes a mutation (thymine to cytosine) at position of 3790 (in a square) that yields C1245R.

The thyroid gland of patient A, removed by surgery, was diffusely enlarged (380 g in weight) with several scattered cystic lesions, having the gross appearance of an adenomatous goiter. As reported (10), an occult papillary carcinoma was found. Microscopically, most of the follicular lumens were dilated to variable degrees (data not shown). Immunohistochemistry demonstrated the presence of Tg in follicular cells but none in the follicular lumen (Fig. 2A). Electron microscopy showed dilated RER within the follicular cell. These findings are consistent with those observed in a case of defective intracellular transport of Tg (Fig. 2B) (14).

View larger version (80K): [in this window] [in a new window]   FIG. 2. Tg in patient A is not secreted in the follicular lumen and retained in RER. A and B, Tg immunohistochemistry and electron microscopy, respectively, of the thyroid gland of patient A. Note that Tg immunostaining was found only in the follicular cell, whereas it was conspicuously absent in the follicular lumen, and that extremely dilated vesicles of RER are present in the follicular cells. C, Digestion of thyroid tissue extracts by Endo H. Thyroid tissues were obtained from a patient homozygous for Tg C1245R, patient A, and a patient with Graves’ disease that did not contain a mutation in Tg cDNA (Normal). An aliquot of the thyroid extract containing 2 µg Tg was digested with Endo H (0.3 mU/liter) and subjected to 4–15% gradient SDS-PAGE using the Phast System (Pharmacia, Uppsala, Sweden). Undigested Tg or Tg resistant to Endo H migrated as a 330-kDa band, whereas Endo H-sensitive Tg was smaller in size. In the thyroid glands of a patient homozygous for Tg C1245R and patient A, proteins of smaller size than the Tg (either resistant or sensitive to Endo H) were present. These presumably represent molecular chaperones. MW, Molecular weight marker.

Demonstration by the pulse-chase experiment that the G2356R mutation caused defective intracellular transport of Tg

To demonstrate whether the G2356R mutation causes a defect in intracellular transport of Tg as does C1245R, we expressed in HEK293 cells the mutant Tgs, G2356R, and C1245R separately as well as the wild-type Tg. As shown in Fig. 3, normal Tg was detected within the cell up to 6 h of the chase, but little remained after 24 h. Tg accumulated in the medium during 24 h of the chase, indicating that most of the wild-type Tg was secreted to the medium in the 24 h just after its synthesis.

View larger version (91K): [in this window] [in a new window]   FIG. 3. Pulse-chase experiment on Tg synthesized by HEK293 cells. Tgs with C1245R and G2356R mutation and the wild-type Tg were individually expressed in HEK293 cells and labeled with [35S]methionine for 3 h. The labeled Tg was then chased for 1, 6, or 24 h. After the harvest, the Tg in the medium and in the cell lysates was immunoprecipitated with anti-Tg antibody and analyzed by 5–15% gradient SDS-PAGE. A [methyl-14C]methylated protein molecular weight (MW) marker (PerkinElmer) was used. Fluorography was performed using Amplify (Amersham International, Little Chalfont, UK), and signals were analyzed by Fuji Bioimage Analyzer (BAS 5000; Fuji Photo Film, Tokyo, Japan). In the cell lysates (top), all three Tgs were detected (white arrow) during the chase of at least 6 h. On the other hand, in the medium (bottom), only the wild-type Tg (white arrow) was detected. Tgs with either C1245R or G2356R mutations were not detected at any time of the chase, indicating that these mutant Tgs were not secreted into the medium. NIS, Nonimmune serum.

Increased D2 activity in thyroid tissues of patients with defective Tg transport

To explore the mechanism(s) by which patients with defective Tg transport have higher serum T₃ levels relative to those of T₄, D2 activity was measured in the thyroid tissues obtained from patient A, two patients with homozygous C1245R mutation, and one patient with homozygous C1977S mutation. Control tissues were obtained from five normal thyroid glands surgically removed for nonfunctioning thyroid adenomas (three cases) or carcinomas (two cases). D2 activity in thyroid glands of patients with defective Tg transport was 2553 ± 479 fmol/mg microsomal protein·h (mean ± SE; n = 4), whereas the mean activity in normal thyroid tissues was 933 ± 392 fmol/mg microsomal protein·h. Thus, D2 activity in the thyroids with defective Tg transport was significantly higher (P < 0.05) than normal (Fig. 4A). Among the normal thyroid tissues, we found that one from a patient with benign adenoma showed high D2 activity that overlapped with that of patients with defective Tg transport. Interestingly, this patient with thyroid adenoma also had a high serum T₃ level relative to that of T₄, resulting in a high FT₃/FT₄ ratio (Fig. 4B). We therefore correlated the FT₃/FT₄ ratio with the corresponding D2 activity in the thyroid tissues and found a positive correlation (r = 0.839; P < 0.01; Fig. 4B). On the other hand, thyroidal D1 activity in patients with Tg defect was 1194 ± 525 pmol/mg microsomal protein·min (mean ± SE, n = 4) and that in controls was 249 ± 70 pmol/mg microsomal protein·min. There was no significant difference in thyroidal D1 activity between the patients and controls (P = 0.083), nor was there a correlation between thyroidal D1 activity and FT₃/FT₄ ratio (r = 0.371; P = 0.365).

View larger version (36K): [in this window] [in a new window]   FIG. 4. D2 activity is increased at a posttranslational level in the thyroid glands of patients with defective intracellular transport of Tg and a positive correlation between the thyroidal D2 activity and serum FT3/FT4 ratio. A, Thyroidal D2 activities were measured in four patients with defective intracellular transport of Tg (two patients with homozygous C1245R mutation, one with homozygous C1977S mutation, and patient A). For control, D2 activity was also measured in normal thyroid tissues obtained from three patients with nonfunctioning thyroid adenomas and two patients with carcinomas. Results are expressed as mean ± SE fmol/mg microsomal protein·h. B, A positive correlation was observed between FT3/FT4 ratio and thyroidal D2 activity. •, Patients with defective intracellular Tg transport; , controls. One patient homozygous for Tg C1245R is not included because data of serum FT4 and FT3 were not available. C, Northern blot analysis of thyroidal D2 mRNA. D2 mRNA in the thyroid gland of patient A is compared with that in normal thyroid tissues. An autoradiograph is shown on the left. The radioactivity of bands were quantitated by densitometry. The amount of D2 mRNA was corrected for the densities of 28S rRNA and expressed in an arbitrary unit (right).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
In the present study, we identified two missense mutations of the Tg gene (C1245R and G2356R) on different Tg alleles in patient A. Analysis of thyroid tissue obtained from a patient with a homozygous C1245R mutation previously showed that Tg with the C1245R mutation is retained in the ER and not transported to the Golgi apparatus (8). However, the consequences of G2356R remained unknown. Results from immunohistochemistry, electron microscopy, and Endo H digestion on the thyroid gland of patient A are consistent with notion that not only C1245R but also G2356R causes an intracellular transport defect. We therefore performed a pulse-chase experiment using G2356R Tg expressed in HEK293 cells. The results clearly demonstrated that the G2356R Tg as well as the C1245R Tg were normally synthesized but were not secreted at all. Thus, we provide a direct demonstration that the G2356R mutation, like C1245R, causes defective intracellular transport of Tg molecule.

One of the clinical features of patients with defective Tg synthesis and/or its transport is a serum T₃ concentration disproportionately higher compared with T₄. In a review, Medeiros-Neto et al. (2) speculate that rapid hydrolysis of limited amounts of Tg is a possible mechanism providing an adequate amount of T₃ (but not T₄), maintaining serum T₃ levels within normal limits. However, this speculation has not been confirmed. Here we propose that increased thyroidal D2 activity is the possible mechanism explaining the increased plasma T₃ levels because a recent study has suggested that D2 plays an important role in maintaining plasma T₃ levels (13). As a matter of fact, it has been suggested that the increased D2 in the thyroid gland may account for the status of low FT₄ with relatively high circulating T₃ levels in other thyroid diseases, such as follicular carcinoma (21) and Graves’ disease during propylthiouracil treatment (22). Results of the present study clearly demonstrated that D2 activity was markedly increased in the thyroid glands obtained from patients with defective Tg transport as compared with that in normal thyroid tissues. This increase in D2 activity is likely due to posttranslational control because D2 mRNA was not increased in the thyroid gland of patient A. Because the disproportional increases in serum T₃ levels are not limited in patients with defective Tg transport but also observed among the patients with congenital goiter due to mutations in the Tg gene (2), it is highly likely that the abnormality of Tg synthesis and/or transport is associated with increased D2 activity in the thyroid gland, resulting in high serum FT₃ levels relative to the T₄ concentration.

D2 has a very short half-life (<1 h) compared with D1 (>12 h) (23). Thus, the posttranslational control of D2 seems to be important for the control of its activity. Furthermore, T₃ and T₄ can exert suppressive effects on D2 activity by pre- and posttranslational mechanisms, respectively, in vivo (24). This may explain why D2 activity in human thyroid glands is suppressed disproportionately to the high levels in its mRNA because T₄ must exist in very high concentration in normal thyroid tissue (23). On the other hand, it is highly possible that T₄ production is impaired in cases of defective Tg synthesis and/or transport. Actually, serum T₄ levels are low in most cases of defective Tg synthesis (2) as well as in our cases with defective Tg transport (10). Therefore, the lower T₄ contents in their thyroid glands may explain the mechanism by which thyroidal D2 activity is much increased in patients with defective intracellular transport of Tg.

Another possible mechanism of the increased D2 activity in thyroid glands with defective Tg transport is impaired proteolysis of D2 by ubiquitination. D2 is ubiquitinated and the D2-ubiquitin conjugates (Ub-D2) are taken up by a proteasome and then rapidly degraded in cells (25). For this process, two ubiquitin-conjugating enzymes, namely Ubc6p and Ubc7p, are required (26). On the other hand, a recent study showed that the expression of molecular chaperones is increased in the thyroid gland with abnormal Tg due to unfolded protein responses (UPR) (27). In fact, a number of molecular chaperones were demonstrated in thyroid tissues obtained from patient A and a patient homozygous for C1245R (Fig. 2C). It is well known that both Ubc6p and Ubc7p are involved in UPR (reviewed in Ref. 28). We thus assumed that a majority of Ubc6p and Ubc7p are recruited in UPR in patients with defective Tg transport and become deficient for D2 degradation, resulting in increased D2 activity.

In summary, we demonstrate that the Tg G2356R mutation found in patient A produces defective intracellular transport of Tg by expressing the mutant Tg in HEK293 cells and performing a pulse-chase experiment. In thyroid glands of patients with such defective intracellular transport of Tg, D2 activity was significantly increased and correlated with serum FT₃/FT₄ ratio. Therefore, we suggest that the increased thyroidal D2 activity accounts for a relatively high serum FT₃ level with a disproportionately low FT₄ level, which is frequently observed in patients with defective intracellular transport of Tg.

	Acknowledgments

 
Thanks are due to Dr. Samuel Refetoff for his valuable suggestions and careful review of this manuscript and to Dr. Junta Takamatsu for providing us thyroid tissues obtained from patients with defective Tg transport.

	Footnotes

 
This work was supported by Grant-in-Aid for Scientific Research (C) to Y.K. and a Center-of-Excellence grant from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

Present address for Y. Miu.: Hoshigaoka Naika, 113 Inoue-cho, Chikusa-ku, Nagoya 464-0026, Japan.

Present address for C.Y.: Department of Diabetes and Clinical Nutrition, Kyoto University, Graduate School of Medicine, 54 Shogoinkawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan.

Disclosure Statement: The authors have nothing to disclose.

First Published Online January 23, 2007

Abbreviations: D2, Type 2 iodothyronine deiodinase; Endo H, endoglycosidase H; FT₃, free T₃; FT₄, free T₄; LT₄, levothyroxine; RER, rough endoplasmic reticulum; Tg, thyroglobulin; UPR, unfolded protein responses.

Received June 9, 2006.

Accepted January 16, 2007.

	References

Top Abstract Introduction Subjects and Methods Results Discussion References

 

1. van de Graaf SA, Cammenga M, Ponne NJ, Veenboer GJ, Gons MH, Orgiazzi J, de Vijlder JJ, Ris-Stalpers C 1999 The screening for mutations in the thyroglobulin cDNA from six patients with congenital hypothyroidism. Biochimie (Paris) 81:425–432
2. Medeiros-Neto G, Targovnik HM, Vassart G 1993 Defective thyroglobulin synthesis and secretion causing goiter and hypothyroidism. Endocr Rev 14:165–183[Abstract/Free Full Text]
3. de Vijlder JJ 2003 Primary congenital hypothyroidism: defects in iodine pathways. Eur J Endocrinol 149:247–256[Abstract]
4. Vono-Toniolo J, Rivolta CM, Targovnik HM, Medeiros-Neto G, Kopp P 2005 Naturally occurring mutations in the thyroglobulin gene. Thyroid 15:1021–1033[CrossRef][Medline]
5. van de Graaf SA, Ris-Stalpers C, Veenboer GJ, Cammenga M, Santos C, Targovnik HM, de Vijlder JJ, Medeiros-Neto G 1999 A premature stopcodon in thyroglobulin messenger RNA results in familial goiter and moderate hypothyroidism. J Clin Endocrinol Metab 84:2537–2542[Abstract/Free Full Text]
6. Targovnik HM, Medeiros-Neto G, Varela V, Cochaux P, Wajchenberg BL, Vassart G 1993 A nonsense mutation causes human hereditary congenital goiter with preferential production of a 171-nucleotide-deleted thyroglobulin ribonucleic acid messenger. J Clin Endocrinol Metab 77:210–215[Abstract]
7. Ieiri T, Cochaux P, Targovnik HM, Suzuki M, Shimoda S, Perret J, Vassart G 1991 A 3' splice site mutation in the thyroglobulin gene responsible for congenital goiter with hypothyroidism. J Clin Invest 88:1901–1905[Medline]
8. Hishinuma A, Takamatsu J, Ohyama Y, Yokozawa T, Kanno Y, Kuma K, Yoshida S, Matsuura N, Ieiri T 1999 Two novel cysteine substitutions (C1263R and C1995S) of thyroglobulin cause a defect in intracellular transport of thyroglobulin in patients with congenital goiter and the variant type of adenomatous goiter. J Clin Endocrinol Metab 84:1438–1444[Abstract/Free Full Text]
9. Hishinuma A, Fukata S, Nishiyama S, Nishi Y, Oh-Ishi M, Murata Y, Ohyama Y, Matsuura N, Kasai K, Harada S, Kitanaka S, Takamatsu J, Kiwaki K, Ohye H, Uruno T, Tomoda C, Tajima T, Kuma K, Miyauchi A, Ieiri T 2006 Haplotype analysis reveals founder effects of thyroglobulin gene mutations C1058R and C1977S in Japan. J Clin Endocrinol Metab 91:3100–3104[Abstract/Free Full Text]
10. Hishinuma A, Fukata S, Kakudo K, Murata Y, Ieiri T 2005 High incidence of thyroid cancer in long-standing goiters with thyroglobulin mutations. Thyroid 15:1079–1084[CrossRef][Medline]
11. Calvo R, Obregon MJ, Ruiz de Ona C, Escobar del Rey F, Morreale de Escobar G 1990 Congenital hypothyroidism, as studied in rats. Crucial role of maternal thyroxine but not of 3,5,3'-triiodothyronine in the protection of the fetal brain. J Clin Invest 86:889–899[Medline]
12. Bernal J 2002 Action of thyroid hormone in brain. J Endocrinol Invest 25:268–288[Medline]
13. Maia AL, Kim BW, Huang SA, Harney JW, Larsen PR 2005 Type 2 iodothyronine deiodinase is the major source of plasma T₃ in euthyroid humans. J Clin Invest 115:2524–2533[CrossRef][Medline]
14. Hishinuma A, Kasai K, Masawa N, Kanno Y, Arimura M, Shimoda SI, Ieiri T 1998 Missense mutation (C1263R) in the thyroglobulin gene causes congenital goiter with mild hypothyroidism by impaired intracellular transport. Endocr J 45:315–327[Medline]
15. Sambrook J, Fritsch EF, Maniatis T 1989 Molecular cloning, a laboratory manual. 2nd ed. New York: Cold Spring Harbor Laboratory Press
16. Murata Y, Magner JA, Refetoff S 1986 The role of glycosylation in the molecular conformation and secretion of thyroxine-binding globulin. Endocrinology 118:1614–1621[Abstract/Free Full Text]
17. Murata Y, Sueda K, Seo H, Matsui N 1989 Studies on the role of glycosylation for human corticosteroid-binding globulin: comparison with that for thyroxine-binding globulin. Endocrinology 125:1424–1429[Abstract/Free Full Text]
18. Murakami M, Araki O, Hosoi Y, Kamiya Y, Morimura T, Ogiwara T, Mizuma H, Mori M2001 Expression and regulation of type II iodothyronine deiodinase in human thyroid gland. Endocrinology 142:2961–2967
19. Murakami M, Tanaka K, Greer MA 1988 There is a nyctohemeral rhythm of type II iodothyronine 5'-deiodinase activity in rat anterior pituitary. Endocrinology 123:1631–1635[Abstract/Free Full Text]
20. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159[Medline]
21. Kim BW, Daniels GH, Harrison BJ, Price A, Harney JW, Larsen PR, Weetman AP 2003 Overexpression of type 2 iodothyronine deiodinase in follicular carcinoma as a cause of low circulating free thyroxine levels. J Clin Endocrinol Metab 88:594–598[Abstract/Free Full Text]
22. Weetman AP, Shepherdley CA, Mansell P, Ubhi CS, Visser TJ 2003 Thyroid over-expression of type 1 and type 2 deiodinase may account for the syndrome of low thyroxine and increasing triiodothyronine during propylthiouracil treatment. Eur J Endocrinol 149:443–447[Abstract]
23. 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]
24. 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]
25. 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]
26. Botero D, Gereben B, Goncalves C, De Jesus LA, Harney JW, Bianco AC 2002 Ubc6p and ubc7p are required for normal and substrate-induced endoplasmic reticulum-associated degradation of the human selenoprotein type 2 iodothyronine monodeiodinase. Mol Endocrinol 16:1999–2007[Abstract/Free Full Text]
27. Baryshev M, Sargsyan E, Wallin G, Lejnieks A, Furudate S, Hishinuma A, Mkrtchian S 2004 Unfolded protein response is involved in the pathology of human congenital hypothyroid goiter and rat non-goitrous congenital hypothyroidism. J Mol Endocrinol 32:903–920[Abstract]
28. Hampton RY 2002 ER-associated degradation in protein quality control and cellular regulation. Curr Opin Cell Biol 14:476–482[CrossRef][Medline]

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