In Vitro Characterization of Human, Mouse, and Zebrafish MCT8 Orthologues.

Background: Mutations in the thyroid hormone (TH) transporter monocarboxylate transporter 8 (MCT8) cause MCT8 deficiency, characterized by severe intellectual and motor disability and abnormal serum thyroid function tests. Various Mct8 knock-out mouse models as well as mct8 knock-out and knockdown zebrafish models are used as a disease model for MCT8 deficiency. Although important for model eligibility, little is known about the functional characteristics of the MCT8 orthologues in these species. Therefore, we here compared the functional characteristics of mouse (mm) MCT8 and zebrafish (dr) Mct8 to human (hs) MCT8. Methods: We performed extensive transport studies in COS-1 and JEG-3 cells transiently transfected with hsMCT8, drMct8, and mmMCT8. Protein expression levels and subcellular localization were assessed by immunoblotting, surface biotinylation, and immunocytochemistry. Sequence alignment and structural modeling were used to interpret functional differences between the orthologues. Results: hsMCT8, drMct8, and mmMCT8 all facilitated the uptake and efflux of 3,3'-diiodothyronine (3,3'-T2), rT3, triiodothyronine (T3), and thyroxine (T4), although the initial uptake rates of drMct8 were 1.5-4.0-fold higher than for hsMCT8 and mmMCT8. drMct8 exhibited 3-50-fold lower apparent IC50 values than hsMCT8 and mmMCT8 for all tested substrates, and substrate preference of drMct8 (3,3'-T2, T3 > T4 > rT3) differed from hsMCT8 and mmMCT8 (T3 > T4 > rT3, 3,3'-T2). Compared with hsMCT8 and mmMCT8, cis-inhibition studies showed that T3 uptake by drMct8 was inhibited at a lower concentration and by a broader spectrum of TH metabolites. Total and cell surface expression levels of drMct8 and hsMCT8 were equal and both significantly exceeded those of mmMCT8. Structural modeling located most non-conserved residues outside the substrate pore, except for H192 in hsMCT8, which is replaced by a glutamine in drMct8. However, a H192Q substituent of hsMCT8 did not alter its transporter characteristics. Conclusion: Our studies substantiate the eligibility of mice and zebrafish models for human MCT8 deficiency. However, differences in the intrinsic transporter properties of MCT8 orthologues may exist, which should be realized when comparing MCT8 deficiency in different in vivo models. Moreover, our findings may indicate that the protein domains outside the substrate channel may play a role in substrate selection and protein stability.

Role of the Bile Acid Transporter SLC10A1 in Liver Targeting of the Lipid-Lowering Thyroid Hormone Analog Eprotirome.

The thyroid hormone (TH) analog eprotirome (KB2115) was developed to lower cholesterol through selective activation of the TH receptor (TR) ß1 in the liver. Interestingly, eprotirome shows low uptake in nonhepatic tissues, explaining its lipid-lowering action without adverse extrahepatic thyromimetic effects. Clinical trials have shown marked decreases in serum cholesterol levels. We explored the transport of eprotirome across the plasma membrane by members of three TH transporter families: monocarboxylate transporters MCT8 and MCT10; Na-independent organic anion transporters 1A2, 1B1, 1B3, 1C1, 2A1, and 2B1; and Na-dependent organic anion transporters SLC10A1 to SLC10A7. Cellular transport was studied in transfected COS1 cells using [14C]eprotirome and [125I]TH analogs. Of the 15 transporters tested initially, the liver-specific bile acid transporter SLC10A1 showed the highest eprotirome uptake (greater than a sevenfold induction after 60 minutes) as well as TRß1-mediated transcriptional activity. Uptake of eprotirome by SLC10A1 was Na+ dependent and saturable with a Michaelis constant of 8 µM. Eprotirome transport was inhibited by known substrates for SLC10A1 (e.g., cholate and taurocholate), and by TH analogs such as triiodothyropropionic acid and triiodothyroacetic acid. However, no significant SLC10A1-mediated transport was observed of these [125I]TH analogs. We also studied the plasma disappearance and biliary excretion of [14C]eprotirome injected in control and Slc10a1 knockout mice. Although eprotirome is also transported by mouse Slc10a1, the pharmacokinetics of eprotirome were not affected by Slc10a1 deficiency. In conclusion, we have demonstrated that the liver-specific bile acid transporter SLC10A1 effectively transports eprotirome. However, Slc10a1 does not appear to be critical for the liver targeting of this TH analog in mice. Therefore, the importance of SLC10A1 for liver uptake of eprotirome in humans remains to be elucidated.

Characterization of Chicken Thyroid Hormone Transporters.

Thyroid hormone (TH) transmembrane transporters are key regulators of TH availability in target cells where correct TH signaling is essential for normal development. Although the chicken embryo is a valuable model for developmental studies, the only functionally characterized chicken TH transporter so far is the organic anion transporting polypeptide 1C1 (OATP1C1). We therefore cloned the chicken L-type amino acid transporter 1 (LAT1) and the monocarboxylate transporters 8 (MCT8) and 10 (MCT10), and functionally characterized them, together with OATP1C1, in JEG3, COS1, and DF-1 cells. In addition, we used in situ hybridization to study their mRNA expression pattern during development. MCT8 and OATP1C1 are both high affinity transporters for the prohormone T4, whereas receptor-active T3 is preferably transported by MCT8 and MCT10. The latter one shows lower affinity but has a high Vmax and seems to be especially good at T3 export. Also, LAT1 has a lower affinity for its preferred substrate 3,3'-diiodothyronine. Reverse T3 is transported by all 4 TH transporters and is a good export product for OATP1C1. TH transporters are strongly expressed in eye (LAT1, MCT8, MCT10), pancreas (LAT1, MCT10), kidney, and testis (MCT8). Their extensive expression in the central nervous system, especially at the brain barriers, indicates an important role in brain development. In conclusion, we show TH transport by chicken MCT8, MCT10, and LAT1. Together with OATP1C1, these transporters have functional characteristics similar to their mammalian orthologs and are interesting target genes to further elucidate the role of THs during embryonic development.

Further Insights into the Allan-Herndon-Dudley Syndrome: Clinical and Functional Characterization of a Novel MCT8 Mutation.

BACKGROUND: Mutations in the thyroid hormone (TH) transporter MCT8 have been identified as the cause for Allan-Herndon-Dudley Syndrome (AHDS), characterized by severe psychomotor retardation and altered TH serum levels. Here we report a novel MCT8 mutation identified in 4 generations of one family, and its functional characterization. METHODS: Proband and family members were screened for 60 genes involved in X-linked cognitive impairment and the MCT8 mutation was confirmed. Functional consequences of MCT8 mutations were studied by analysis of [125I]TH transport in fibroblasts and transiently transfected JEG3 and COS1 cells, and by subcellular localization of the transporter. RESULTS: The proband and a male cousin demonstrated clinical findings characteristic of AHDS. Serum analysis showed high T3, low rT3, and normal T4 and TSH levels in the proband. A MCT8 mutation (c.869C>T; p.S290F) was identified in the proband, his cousin, and several female carriers. Functional analysis of the S290F mutant showed decreased TH transport, metabolism and protein expression in the three cell types, whereas the S290A mutation had no effect. Interestingly, both uptake and efflux of T3 and T4 was impaired in fibroblasts of the proband, compared to his healthy brother. However, no effect of the S290F mutation was observed on TH efflux from COS1 and JEG3 cells. Immunocytochemistry showed plasma membrane localization of wild-type MCT8 and the S290A and S290F mutants in JEG3 cells. CONCLUSIONS: We describe a novel MCT8 mutation (S290F) in 4 generations of a family with Allan-Herndon-Dudley Syndrome. Functional analysis demonstrates loss-of-function of the MCT8 transporter. Furthermore, our results indicate that the function of the S290F mutant is dependent on cell context. Comparison of the S290F and S290A mutants indicates that it is not the loss of Ser but its substitution with Phe, which leads to S290F dysfunction.

In vitro and mouse studies supporting therapeutic utility of triiodothyroacetic acid in MCT8 deficiency.

Monocarboxylate transporter 8 (MCT8) transports thyroid hormone (TH) across the plasma membrane. Mutations in MCT8 result in the Allan-Herndon-Dudley syndrome, comprising severe psychomotor retardation and elevated serum T3 levels. Because the neurological symptoms are most likely caused by a lack of TH transport into the central nervous system, the administration of a TH analog that does not require MCT8 for cellular uptake may represent a therapeutic strategy. Here, we investigated the therapeutic potential of the biologically active T3 metabolite Triac (TA3) by studying TA3 transport, metabolism, and action both in vitro and in vivo. Incubation of SH-SY5Y neuroblastoma cells and MO3.13 oligodendrocytes with labeled substrates showed a time-dependent uptake of T3 and TA3. In intact SH-SY5Y cells, both T3 and TA3 were degraded by endogenous type 3 deiodinase, and they influenced gene expression to a similar extent. Fibroblasts from MCT8 patients showed an impaired T3 uptake compared with controls, whereas TA3 uptake was similar in patient and control fibroblasts. In transfected cells, TA3 did not show significant transport by MCT8. Most importantly, treatment of athyroid Pax8-knockout mice and Mct8/Oatp1c1-double knockout mice between postnatal days 1 and 12 with TA3 restored T3-dependent neural differentiation in the cerebral and cerebellar cortex, indicating that TA3 can replace T3 in promoting brain development. In conclusion, we demonstrated uptake of TA3 in neuronal cells and in fibroblasts of MCT8 patients and similar gene responses to T3 and TA3. This indicates that TA3 bypasses MCT8 and may be used to improve the neural status of MCT8 patients.

Psychomotor retardation caused by a defective thyroid hormone transporter: report of two families with different MCT8 mutations.

BACKGROUND/AIMS: Monocarboxylate transporter 8 (MCT8) is essential for thyroid hormone (TH) transport in the brain. Mutations in MCT8 are associated with the Allan-Herndon-Dudley syndrome (AHDS), characterized by severe psychomotor retardation and altered serum thyroid parameters. Here we report two novel mutations in MCT8 and discuss the clinical findings. CASE REPORT AND RESULTS: We describe 4 males with AHDS from two unrelated families varying in age from 1.5 to 11 years. All 4 patients presented with typical clinical signs of AHDS, including severe psychomotor retardation, axial hypotonia, lack of speech, diminished muscle mass, increased muscle tone, hyperreflexia, myopathic facies, high T3, low T4 and rT3, and normal/mildly elevated TSH levels. Comparison of patients at different ages suggests the progressive nature of AHDS. Genetic analyses identified a novel missense MCT8 mutation (p.G495A) in family 1 and a 2.8-kb deletion comprising exons 3 and 4 in family 2. Functional analysis of p.G495A revealed impaired TH transport varying from 20 to 85% depending on the cell context. CONCLUSION: Here we report 4 AHDS patients in unrelated Turkish families harboring novel MCT8 mutations. Despite the widely different mutations, the clinical phenotypes are very similar and findings support the progressive nature of AHDS.

Knockdown of type 3 iodothyronine deiodinase severely perturbs both embryonic and early larval development in zebrafish.

Exposure to appropriate levels of thyroid hormones (THs) at the right time is of key importance for normal development in all vertebrates. Type 3 iodothyronine deiodinase (D3) is the prime TH-inactivating enzyme, and its expression is highest in the early stages of vertebrate development, implying that it may be necessary to shield developing tissues from overexposure to THs. We used antisense morpholino knockdown to examine the role of D3 during early development in zebrafish. Zebrafish possess 2 D3 genes, dio3a and dio3b. Here, we show that both genes are expressed during development and both contribute to in vivo D3 activity. However, dio3b mRNA levels in embryos are higher, and the effects of dio3b knockdown on D3 activity and on the resulting phenotype are more severe. D3 knockdown induced an overall delay in development, as determined by measurements of otic vesicle length, eye and ear size, and body length. The time of hatching was also severely delayed in D3-knockdown embryos. Importantly, we also observed a severe disturbance of several aspects of development. Swim bladder development and inflation was aberrant as was the development of liver and intestine. Furthermore, D3-knockdown larvae spent significantly less time moving, and both embryos and larvae exhibited perturbed escape responses, suggesting that D3 knockdown affects muscle development and/or functioning. These data indicate that D3 is essential for normal zebrafish embryonic and early larval development and show the value of morpholino knockdown in this model to further elucidate the specific role of D3 in some aspects of vertebrate development.

The role of Arg445 and Asp498 in the human thyroid hormone transporter MCT8.

Monocarboxylate transporter 8 (MCT8) facilitates cellular influx and efflux of the thyroid hormones (THs) T(4) and T(3). Mutations in MCT8 lead to severe psychomotor retardation. Here, we studied the importance of 2 highly conserved residues (Arg445 in transmembrane domain 8 and Asp498 in transmembrane domain 10) for substrate recognition and helix interactions. We introduced single and double mutations (R445A, R445C, R445D, R445K, D498A, D498E, D498N, D498R, R445A+D498A, R445D+D498R, and R445K+D498E) in human MCT8 cDNA and studied the effects on MCT8-mediated TH uptake and metabolism in transfected cells. The impact of these mutations on MCT8 protein expression, dimerization capacity, and subcellular localization was studied by Western blotting and confocal microscopy. We found that mutations in Arg445 or Asp498 that alter the local charge resulted in a near-complete loss of TH uptake capacity, whereas the expression, stability, and subcellular localization of these mutant proteins was similar to those for wild-type MCT8. Given the impaired TH uptake, TH efflux could not be adequately studied. The importance of opposite charges at Arg445 and Asp498 was studied by exchanging these residues (R445D+D498R). In particular, T(4) uptake was less severely reduced by the exchange mutation than by the single mutations. Mutations of Arg445 and Asp498 to equally charged residues (R445K and/or D498E) resulted in TH uptake levels similar to wild-type MCT8. The presence of 2 oppositely charged residues at positions Arg445 and Asp498 that are predicted in close structural proximity is crucial for efficient TH uptake, which may indicate the presence of an, at least transient, charge pair between these residues.

Mutations in MCT8 in patients with Allan-Herndon-Dudley-syndrome affecting its cellular distribution.

Monocarboxylate transporter 8 (MCT8) is a thyroid hormone (TH)-specific transporter. Mutations in the MCT8 gene are associated with Allan-Herndon-Dudley Syndrome (AHDS), consisting of severe psychomotor retardation and disturbed TH parameters. To study the functional consequences of different MCT8 mutations in detail, we combined functional analysis in different cell types with live-cell imaging of the cellular distribution of seven mutations that we identified in patients with AHDS. We used two cell models to study the mutations in vitro: 1) transiently transfected COS1 and JEG3 cells, and 2) stably transfected Flp-in 293 cells expressing a MCT8-cyan fluorescent protein construct. All seven mutants were expressed at the protein level and showed a defect in T3 and T4 transport in uptake and metabolism studies. Three mutants (G282C, P537L, and G558D) had residual uptake activity in Flp-in 293 and COS1 cells, but not in JEG3 cells. Four mutants (G221R, P321L, D453V, P537L) were expressed at the plasma membrane. The mobility in the plasma membrane of P537L was similar to WT, but the mobility of P321L was altered. The other mutants studied (insV236, G282C, G558D) were predominantly localized in the endoplasmic reticulum. In essence, loss of function by MCT8 mutations can be divided in two groups: mutations that result in partial or complete loss of transport activity (G221R, P321L, D453V, P537L) and mutations that mainly disturb protein expression and trafficking (insV236, G282C, G558D). The cell type-dependent results suggest that MCT8 mutations in AHDS patients may have tissue-specific effects on TH transport probably caused by tissue-specific expression of yet unknown MCT8-interacting proteins.

Relevance of different cellular models in determining the effects of mutations on SLC16A2/MCT8 thyroid hormone transporter function and genotype-phenotype correlation.

SLC 16A2, the gene for the second member of the solute carrier family 16 (monocarboxylic acid transporter), located on chromosome Xq13.2, encodes a very efficient thyroid hormone transporter: monocarboxylate transporter 8, MCT8. Its loss of function is responsible in males for a continuum of psychomotor retardation ranging from severe (no motor acquisition, no speech) to mild (ability to walk with help and a few words of speech). Triiodothyronine uptake measurement in transfected cells and, more recently, patient fibroblasts, has been described to study the functional consequences of MCT8 mutations. Here, we describe three novel MCT8 mutations, including one missense variation not clearly predicted to be damaging but found in a severely affected patient. Functional studies in fibroblasts and JEG3 cells demonstrate the usefulness of both cellular models in validating the deleterious effects of a new MCT8 mutation if there is still a doubt as to its pathogenicity. Moreover, the screening of fibroblasts from a large number of patient fibroblasts and of transfected mutations has allowed us to demonstrate that JEG3 transfected cells are more relevant than fibroblasts in revealing a genotype-phenotype correlation.

Tetrac can replace thyroid hormone during brain development in mouse mutants deficient in the thyroid hormone transporter mct8.

The monocarboxylate transporter 8 (MCT8) plays a critical role in mediating the uptake of thyroid hormones (THs) into the brain. In patients, inactivating mutations in the MCT8 gene are associated with a severe form of psychomotor retardation and abnormal serum TH levels. Here, we evaluate the therapeutic potential of the TH analog 3,5,3',5'-tetraiodothyroacetic acid (tetrac) as a replacement for T(4) in brain development. Using COS1 cells transfected with TH transporter and deiodinase constructs, we could show that tetrac, albeit not being transported by MCT8, can be metabolized to the TH receptor active compound 3,3',5-triiodothyroacetic acid (triac) by type 2 deiodinase and inactivated by type 3 deiodinase. Triac in turn is capable of replacing T(3) in primary murine cerebellar cultures where it potently stimulates Purkinje cell development. In vivo effects of tetrac were assessed in congenital hypothyroid Pax8-knockout (KO) and Mct8/Pax8 double-KO mice as well as in Mct8-KO and wild-type animals after daily injection of tetrac (400 ng/g body weight) during the first postnatal weeks. This treatment was sufficient to promote TH-dependent neuronal differentiation in the cerebellum, cerebral cortex, and striatum but was ineffective in suppressing hypothalamic TRH expression. In contrast, TSH transcript levels in the pituitary were strongly down-regulated in response to tetrac. Based on our findings we propose that tetrac administration offers the opportunity to provide neurons during the postnatal stage with a potent TH receptor agonist, thereby eventually reducing the neurological damage in patients with MCT8 mutations without deteriorating the thyrotoxic situation in peripheral tissues.