Jurgen Jansen

Effective Cellular Uptake and Efflux of Thyroid Hormone by Human Monocarboxylate Transporter 10

Cellular entry of thyroid hormone is mediated by plasma membrane transporters, among others a T-type (aromatic) amino acid transporter. Monocarboxylate transporter 10 (MCT10) has been reported to transport aromatic amino acids but not iodothyronines. Within the MCT family, MCT10 is most homologous to MCT8, which is a very important iodothyronine transporter but does not transport amino acids. In view of this paradox, we decided to reinvestigate the possible transport of thyroid hormone by human (h) MCT10 in comparison with hMCT8. Transfection of COS1 cells with hMCT10 cDNA resulted in 1) the production of an approximately 55 kDa protein located to the plasma membrane as shown by immunoblotting and confocal microscopy, 2) a strong increase in the affinity labeling of intracellular type I deiodinase by N-bromoacetyl-[(125)I]T(3), 3) a marked stimulation of cellular T(4) and, particularly, T(3) uptake, 4) a significant inhibition of T(3) uptake by phenylalanine, tyrosine, and tryptophan of 12.5%, 22.2%, and 51.4%, respectively, and 5) a marked increase in the intracellular deiodination of T(4) and T(3) by different deiodinases. Cotransfection studies using the cytosolic thyroid hormone-binding protein micro-crystallin (CRYM) indicated that hMCT10 facilitates both cellular uptake and efflux of T(4) and T(3). In the absence of CRYM, hMCT10 and hMCT8 increased T(3) uptake after 5 min incubation up to 4.0- and 1.9-fold, and in the presence of CRYM up to 6.9- and 5.8-fold, respectively. hMCT10 was less active toward T(4) than hMCT8. These findings establish that hMCT10 is at least as active a thyroid hormone transporter as hMCT8, and that both transporters facilitate iodothyronine uptake as well as efflux.

Thyroid hormone transport in and out of cells

Thyroid hormone (TH) is essential for the proper development of numerous tissues, notably the brain. TH acts mostly intracellularly, which requires transport by TH transporters across the plasma membrane. Although several transporter families have been identified, only monocarboxylate transporter (MCT)8, MCT10 and organic anion-transporting polypeptide (OATP)1C1 demonstrate a high degree of specificity towards TH. Recently, the biological importance of MCT8 has been elucidated. Mutations in MCT8 are associated with elevated serum T(3) levels and severe psychomotor retardation, indicating a pivotal role for MCT8 in brain development. MCT8 knockout mice lack neurological damage, but mimic TH abnormalities of MCT8 patients. The exact pathophysiological mechanisms in MCT8 patients remain to be elucidated fully. Future research will probably identify novel TH transporters and disorders based on TH transporter defects.

Mechanisms of disease: psychomotor retardation and high T3 levels caused by mutations in monocarboxylate transporter 8.

The actions and the metabolism of thyroid hormone are intracellular events that require the transport of iodothyronines across the plasma membrane. It is increasingly clear that this process does not occur by simple diffusion, but is facilitated by transport proteins. Only recently have iodothyronine transporters been identified at the molecular level, of which organic anion transporting polypeptide 1C1 and monocarboxylate transporter 8 (MCT8) deserve special mention, because of their high activity and specificity for iodothyronines. Organic anion transporting polypeptide 1C1 is almost exclusively expressed in brain capillaries, and may be crucial for the transport of the prohormone T4 across the blood–brain barrier. MCT8 is also expressed in the brain—in particular, in neurons—but also in other tissues. MCT8 seems to be especially important for the uptake of active hormone T3 into neurons, which is essential for optimal brain development. T3 is produced from T4 by type 2 deiodinase in neighboring astrocytes. Neurons express type 3 deiodinase, the enzyme that terminates T3 activity. The SLC16A2 (formerly MCT8) gene is located on chromosome Xq13.2 and has recently been associated with a syndrome combining severe, X-linked, psychomotor retardation and high serum T3 levels. In over 20 families, where affected males have developed this syndrome, several mutations in MCT8 have been identified. The disease mechanism is thought to involve a defect in the neuronal entry of T3 and, therefore, in the action and metabolism of T3 in these cells. This defect results in impaired neurological development and a decrease in T3 clearance.

Thyroid hormone transporters

Thyroid hormone is essential for the development of the brain and the nervous system. Cellular entry is required for conversion of thyroid hormones by the intracellular deiodinases and for binding of T(3) to its nuclear receptors. Several transporters capable of thyroid hormone transport have been identified. Functional expression studies using Xenopus laevis oocytes have so far identified two categories of transporters involved in thyroid hormone uptake (i.e., organic anion transporters and amino acid transporters). Among the organic anion transporters, both Na(+) taurocholate cotransporting polypeptide (NTCP) and various members of the organic anion transporting polypeptide (OATP) family mediate transport of iodothyronines. Because iodothyronines are a particular class of amino acids derived from tyrosine residues, it is no surprise that some amino acid transporters have been shown to be involved in thyroid hormone transport. We have characterized monocarboxylate transporter 8 (MCT8) as a very active and specific thyroid hormone transporter, the gene of which is located on the X chromosome. MCT8 is highly expressed in liver and brain but is also widely distributed in other tissues. MCT8 shows 50% amino acid identity with a system T amino acid transporter 1 (TAT1). TAT1, also called MCT10, has been characterized to transport aromatic amino acids but no iodothyronines. We have also found that mutations in MCT8 are associated with severe X-linked psychomotor retardation and strongly elevated serum T(3) levels in young boys.

Genotype-phenotype relationship in patients with mutations in thyroid hormone transporter MCT8

Loss-of-function mutations in thyroid hormone transporter monocarboxylate transporter 8 (MCT8) lead to severe X-linked psychomotor retardation and elevated serum T(3) levels. Most patients, for example those with mutations V235M, S448X, insI189, or delF230, cannot stand, walk, or speak. Patients with mutations L434W, L568P, and S194F, however, walk independently and/or develop some dysarthric speech. To study the relationship between mutation and phenotype, we transfected JEG3 and COS1 cells with wild-type or mutant MCT8. Expression and function of the transporter were studied by analyzing T(3) and T(4) uptake, T(3) metabolism (by cotransfected type 3 deiodinase), Western blotting, affinity labeling with N-bromoacetyl-T(3), immunocytochemistry, and quantitative RT-PCR. Wild-type MCT8 increased T(3) uptake and metabolism about 5-fold compared with empty vector controls. Mutants V235M, S448X, insI189, and delF230 did not significantly increase transport. However, S194F, L568P, and L434W showed about 20, 23, and 37% of wild-type activity. RT-PCR did not show significant differences in mRNA expression between wild-type and mutant MCT8. Immunocytochemistry detected the nonfunctional mutants V235M, insI189, and delF230 mostly in the cytoplasm, whereas mutants with residual function were expressed at the plasma membrane. Mutants S194F and L434W showed high protein expression but low affinity for N-bromoacetyl-T(3); L568P was detected in low amounts but showed relatively high affinity. Mutations in MCT8 cause loss of function through reduced protein expression, impaired trafficking to the plasma membrane, or reduced substrate affinity. Mutants L434W, L568P, and S194F showed significant residual transport capacity, which may underlie the more advanced psychomotor development observed in patients with these mutations.

Beneficial effects of propylthiouracil plus L-thyroxine treatment in a patient with a mutation in MCT8

CONTEXT Mutations of the monocarboxylate transporter 8 (MCT8) gene determine a distinct X-linked phenotype of severe psychomotor retardation and consistently elevated T(3) levels. Lack of MCT8 transport of T(3) in neurons could explain the neurological phenotype. OBJECTIVE Our objective was to determine whether the high T(3) levels could also contribute to some critical features observed in these patients. RESULTS A 16-yr-old boy with severe psychomotor retardation and hypotonia was hospitalized for malnutrition (body weight = 25 kg) and delayed puberty. He had tachycardia (104 beats/min), high SHBG level (261 nmol/liter), and elevated serum free T(3) (FT(3)) level (11.3 pmol/liter), without FT(4) and TSH abnormalities. A missense mutation of the MCT8 gene was present. Oral overfeeding was unsuccessful. The therapeutic effect of propylthiouracil (PTU) and then PTU plus levothyroxine (LT(4)) was tested. After PTU (200 mg/d), serum FT(4) was undetectable, FT(3) was reduced (3.1 pmol/liter) with high TSH levels (50.1 mU/liter). Serum SHBG levels were reduced (72 nmol/liter). While PTU prescription was continued, high LT(4) doses (100 microg/d) were needed to normalize serum TSH levels (3.18 mU/liter). At that time, serum FT(4) was normal (16.4 pmol/liter), and FT(3) was slightly high (6.6 pmol/liter). Tachycardia was abated (84 beats/min), weight gain was 3 kg in 1 yr, and SHBG was 102 nmol/liter. CONCLUSIONS 1) When thyroid hormone production was reduced by PTU, high doses of LT(4) (3.7 microg/kg.d) were needed to normalize serum TSH, confirming that mutation of MCT8 is a cause of resistance to thyroid hormone. 2) High T(3) levels might exhibit some deleterious effects on adipose, hepatic, and cardiac levels. 3) PTU plus LT(4) could be an effective therapy to reduce general adverse features, unfortunately without benefit on the psychomotor retardation.

Decreased cellular uptake and metabolism in Allan-Herndon-Dudley syndrome (AHDS) due to a novel mutation in the MCT8 thyroid hormone transporter

We report a novel 1 bp deletion (c.1834delC) in the MCT8 gene in a large Brazilian family with Allan-Herndon-Dudley syndrome (AHDS), an X linked condition characterised by severe mental retardation and neurological dysfunction. The c.1834delC segregates with the disease in this family and it was not present in 100 control chromosomes, further confirming its pathogenicity. This mutation causes a frameshift and the inclusion of 64 additional amino acids in the C-terminal region of the protein. Pathogenic mutations in the MCT8 gene, which encodes a thyroid hormone transporter, results in elevated serum triiodothyronine (T3) levels, which were confirmed in four affected males of this family, while normal levels were found among obligate carriers. Through in vitro functional assays, we showed that this mutation decreases cellular T3 uptake and intracellular T3 metabolism. Therefore, the severe neurological defects present in the patients are due not only to deficiency of intracellular T3, but also to altered metabolism of T3 in central neurones. In addition, the severe muscle hypoplasia observed in most AHDS patients may be a consequence of high serum T3 levels.

Novel pathogenic mechanism suggested by ex vivo analysis of MCT8 (SLC16A2) mutations

Monocarboxylate transporter 8 (MCT8; approved symbol SLC16A2) facilitates cellular uptake and efflux of 3,3′,5‐triiodothyronine (T3). Mutations in MCT8 are associated with severe psychomotor retardation, high serum T3 and low 3,3′,5′‐triiodothyronine (rT3) levels. Here we report three novel MCT8 mutations. Two subjects with the F501del mutation have mild psychomotor retardation with slightly elevated T3 and normal rT3 levels. T3 uptake was mildly affected in F501del fibroblasts and strongly decreased in fibroblasts from other MCT8 patients, while T3 efflux was always strongly reduced. Moreover, type 3 deiodinase activity was highly elevated in F501del fibroblasts, whereas it was reduced in fibroblasts from other MCT8 patients, probably reflecting parallel variation in cellular T3 content. Additionally, T3‐responsive genes were markedly upregulated by T3 treatment in F501del fibroblasts but not in fibroblasts with other MCT8 mutations. In conclusion, mutations in MCT8 result in a decreased T3 uptake in skin fibroblasts. The much milder clinical phenotype of patients with the F501del mutation may be correlated with the relatively small decrease in T3 uptake combined with an even greater decrease in T3 efflux. If fibroblasts are representative of central neurons, abnormal brain development associated with MCT8 mutations may be the consequence of either decreased or increased intracellular T3 concentrations. Hum Mutat 0,1‐10, 2008.

Evidence for a homodimeric structure of human monocarboxylate transporter 8.

The human monocarboxylate transporter 8 (hMCT8) protein mediates transport of thyroid hormone across the plasma membrane. Association of hMCT8 mutations with severe psychomotor retardation and disturbed thyroid hormone levels has established its physiological relevance, but little is still known about the basic properties of hMCT8. In this study we present evidence that hMCT8 does not form heterodimers with the ancillary proteins basigin, embigin, or neuroplastin, unlike other MCTs. In contrast, it is suggested that MCT8 exists as monomer and homodimer in transiently and stably transfected cells. Apparently hMCT8 forms stable dimers because the complex is resistant to denaturing conditions and dithiothreitol. Cotransfection of wild-type hMCT8 with a mutant lacking amino acids 267-360 resulted in formation of homo-and heterodimers of the variants, indicating that transmembrane domains 4-6 are not involved in the dimerization process. Furthermore, we explored the structural and functional role of the 10 Cys residues in hMCT8. All possible Cys>Ala mutants did not behave differently from wild-type hMCT8 in protein expression, cross-linking experiments with HgCl(2) and transport function. Our findings indicate that individual Cys residues are not important for the function of hMCT8 or suggest that hMCT8 has other yet-undiscovered functions in which cysteines play an essential role.