Thyroid hormone transport by the human monocarboxylate transporter 8 and its rate-limiting role in intracellular metabolism.

Cellular entry of thyroid hormone is mediated by plasma membrane transporters. We have identified rat monocarboxylate transporter 8 (MCT8) as an active and specific thyroid hormone transporter. The MCT8 gene is located on the X-chromosome. The physiological relevance of MCT8 has been demonstrated by the identification of hemizygous mutations in this gene in males with severe psychomotor retardation and elevated serum T(3) levels. We have characterized human (h) MCT8 by analysis of iodothyronine uptake and metabolism in cell lines transiently transfected with hMCT8 cDNA alone or together with cDNA coding for iodothyronine deiodinase D1, D2, or D3. MCT8 mRNA was detected by RT-PCR in a number of human cell lines as well as in COS1 cells but was low to undetectable in other cell lines, including JEG3 cells. MCT8 protein was not detected in nontransfected cell lines tested by immunoblotting using a polyclonal C-terminal hMCT8 antibody but was detectable in transfected cells at the expected size (61 kDa). Transfection of COS1 and JEG3 cells with hMCT8 cDNA resulted in 2- to 3-fold increases in uptake of T(3) and T(4) but little or no increase in rT(3) or 3,3'-diiodothyronine (3,3'-T(2)) uptake. MCT8 expression produced large increases in T(4) metabolism by cotransfected D2 or D3, T(3) metabolism by D3, rT(3) metabolism by D1 or D2, and 3,3'-T(2) metabolism by D3. Affinity labeling of hMCT8 protein was observed after incubation of intact transfected cells with N-bromoacetyl-[(125)I]T(3). hMCT8 also facilitated affinity labeling of cotransfected D1 by bromoacetyl-T(3). Our findings indicate that hMCT8 mediates plasma membrane transport of iodothyronines, thus increasing their intracellular availability.

Regulation of type III iodothyronine deiodinase expression in human cell lines.

Type I iodothyronine deiodinase (D1) and type II iodothyronine deiodinase (D2) catalyze the activation of the prohormone T4 to the active hormone T3; type III iodothyronine deiodinase (D3) catalyzes the inactivation of T4 and T3. D3 is highly expressed in brain, placenta, pregnant uterus, and fetal tissues and plays an important role in regulating thyroid hormone bioavailability during fetal development. We examined the activity of the different deiodinases in human cell lines and investigated the regulation of D3 activity and mRNA expression in these cell lines, as well as its possible coexpression with neighboring genes Dlk1 and Dio3os, which may also be especially important during development. D1 activity and mRNA were only found in HepG2 hepatocarcinoma cells, and D2 activity was observed in none of the cell lines. D3 activity and mRNA was found in ECC-1 endometrium carcinoma cells, MCF-7 mammacarcinoma cells, WRL-68 embryonic liver cells, and SH-SY5Y neuroblastoma cells, but not in the HepG2 hepatocarcinoma cell line or in any choriocarcinoma or astrocytoma cell line. We demonstrated that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate increased D3 activity 2- to 9-fold in ECC-1, MCF-7, WRL-68, and SH-SY5Y cells. Estradiol increased D3 activity 3-fold in ECC-1, but not in any other cells. Dexamethasone decreased D3 activity in WRL-68 cells only in the absence of fetal calf serum. Incubation with retinoids increased D3 activity 2- to 3-fold in ECC-1, WRL-68, and MCF-7 cells but decreased D3 activity in SH-SY5Y cells. D3 expression in the different cells was not affected by cAMP or thyroid hormone. Interestingly, D3 mRNA expression in the different cell lines strongly correlated with Dio3os mRNA expression and in a large set of neuroblastoma cell lines also with Dlk1 expression. In conclusion, we identified different human D3-expressing cell lines, in which the regulation of D3 expression is cell type-specific. Our data suggest that estradiol may be one of the factors contributing to the induction of D3 activity in the pregnant uterus and that in addition to gene-specific regulatory elements, more distant common regulatory elements also may be involved in the regulation of D3 expression.

Novel neuroanatomical pathways for thyroid hormone action in the human anterior pituitary.

OBJECTIVE: An increasing number of proteins appear to be involved in thyroid hormone feedback action at the level of the anterior pituitary, but the cell types expressing these proteins are largely unknown. The aim of the present study was to identify cell types in the human anterior pituitary that express type II and type III deiodinase (D2 and D3), the recently described thyroid hormone transporter (MCT8) and thyroid hormone receptor (TR) isoforms by means of double-labeling immunocytochemistry. RESULTS: We found TR isoforms to be expressed most prominently in gonadotropes and - although to a lesser extent - in thyrotropes, corticotropes, lactotropes and somatotropes. D3 staining showed a distribution pattern that was remarkably similar. By contrast, D2 immunoreactivity was observed exclusively in folliculostellate (FS) cells showing coexpression with human leukocyte antigen (HLA), a marker of major histocompatibility complex (MHC)-class II. MCT8 immunostaining was present in FS cells without HLA coexpression. CONCLUSIONS: From these results, we propose a novel neuroanatomical model for thyroid hormone feedback on the human pituitary, with a central role for FS cells in thyroid hormone activation, which thus play an important role in the suppression of TSH secretion by circulating thyroxine (T(4)).

Expression of recombinant membrane-bound type I iodothyronine deiodinase in yeast.

The bioactivity of thyroid hormone is determined to a large extent by the monodeiodination of the prohormone thyroxine (T4) by the hepatic selenoenzyme type I iodothyronine deiodinase (D1), i.e. by outer ring deiodination (ORD) to the active hormone triiodothyronine (T3) or by inner ring deiodination (IRD) to the inactive metabolite reverse T3 (rT3). Since D1 is a membrane-bound protein with an N-terminal membrane-spanning domain, the enzyme is very difficult to purify in an active state. This study was undertaken in order to develop a heterologous (over)-expression system that would eventually allow the production of large amounts of purified active D1 protein. We have expressed a mutant rat D1 protein, in which the selenocysteine residue in the core catalytic center was replaced by cysteine (D1 Cys) in yeast cells (Saccharomyces cerevisiae). After yeast cell fractionation, kinetic analysis was performed with dithiothreitol as reducing cofactor. ORD activity was associated with membrane fractions, while no activity could be detected in the cytosolic fraction. The D1 Cys protein displayed a tenfold increase in Km (2 microM) for rT3 as compared with native D1 protein in rat liver microsomes. The D1 protein content is about 65 pmol/mg microsomal protein, as compared with about 3 pmol/mg in rat liver microsomal fraction. SDS-PAGE analysis of N-bromoacetyl-[125I]T3 affinity-labeled D1 protein showed several labeled protein isoforms with apparent molecular masses between 27 and 32 kDa. Immunoblot analysis with a specific D1 antiserum confirmed the observed D1 protein heterogeneity. Site-directed mutagenesis of several potential N-linked glycosylation sites, phosphorylation sites and a unique myristoylation site established that D1 heterogeneity is not caused by N-linked glycosylation, but probably by a combination of O-linked glycosylation and phosphorylation. Deletion of the endoplasmic reticulum (ER)-signal sequence and the membrane-spanning domain (amino acid residue 2-35), did not result in the production of a soluble D1 enzyme. Although this mutated D1 protein is inactive, the fact that it is still membrane bound indicates the existence of additional membrane attachment site(s) or membrane-spanning domains. Overall, our studies indicate that yeast cells provide a useful system for the expression of relatively high levels of D1 protein which could be used for further structure-function analysis.

Neuroanatomical pathways for thyroid hormone feedback in the human hypothalamus.

CONTEXT: Recent findings point to an increasing number of hypothalamic proteins involved in the central regulation of thyroid hormone feedback. The functional neuroanatomy of these proteins in the human hypothalamus is largely unknown at present. OBJECTIVE: The aim of this study was to report the distribution of type II and type III deiodinase (D2 and D3) as well as the recently identified T(3) transporter, monocarboxylate transporter 8 (MCT8), in the human hypothalamus. DESIGN: The study included enzyme activity assays, immunocytochemical studies, and mRNA in situ hybridizations in postmortem human hypothalamus (n = 9). RESULTS: D2 immunoreactivity is prominent in glial cells of the infundibular nucleus/median eminence, blood vessels, and cells lining the third ventricle. By contrast, both D3 and MCT8 are expressed by neurons of the paraventricular (PVN), supraoptic, and infundibular nucleus (IFN). In support of these immunocytochemical data, D2 and D3 enzyme activities are detectable in the mediobasal human hypothalamus. Combined D2, D3, MCT8, and thyroid hormone receptor immunohistochemistry and TRH mRNA in situ hybridization clearly showed that D3, MCT8, and thyroid hormone receptor isoforms are all expressed in TRH neurons of the PVN, whereas D2 is not. CONCLUSIONS AND IMPLICATIONS: Based on these findings, we propose three possible routes for thyroid hormone feedback on TRH neurons in the human PVN: 1) local thyroid hormone uptake from the vascular compartment within the PVN, 2) thyroid hormone uptake from the cerebrospinal fluid in the third ventricle followed by transport to TRH neurons in the PVN or IFN neurons projecting to TRH neurons in the PVN, and 3) thyroid hormone sensing in the IFN of the mediobasal hypothalamus by neurons projecting to TRH neurons in the PVN.

An ascidian homolog of vertebrate iodothyronine deiodinases.

In all classes of vertebrates, the deiodination of the prohormone T(4) to T(3) represents an essential activation step in thyroid hormone action. The possible presence of iodothyronine deiodinase activity in protochordates has been demonstrated in vivo. Recent molecular cloning of the genomes and transcripts of several ascidian species allows further investigation into thyroid-related processes in ascidians. A cDNA clone from Halocynthia roretzi (hrDx) was found to have significant homology (30% amino acid identity) with the iodothyronine deiodinase gene sequences from vertebrates, including the presence of an in-frame UGA codon that might encode a selenocysteine (SeC) in the active site. Because it was not certain that the 3' untranslated region (UTR) contained a SeC insertion sequence (SECIS) element essential for SeC incorporation, a chimeric expression vector of the hrDx coding sequence and the rat deiodinase SECIS element was produced, as well as an expression vector containing the intact hrDx cDNA. COS, CHO, and HEK cells were transfected with these vectors, and deiodinase activity was measured in cell homogenates. Outer-ring deiodinase activity was detected using both T(4) and reverse T(3) as substrates, and activity was enhanced by the presence of the reductive cofactor dithiothreitol. The enzyme activity was optimal during incubation between 20 and 30 C (pH 6-7) and was strongly inhibited by gold-thioglucose. The Halocynthia deiodinase appears to be a high Michaelis-Menten constant (K(m)) enzyme (K(m) reverse T(3), 2 microM; and K(m) T(4), 4 microM). Deiodinase activity was completely lost upon the substitution of the SeC residue in the putative catalytic center by either cysteine or alanine. Transfection of the full-length hrDx cDNA produced deiodinase activity confirming the presence of a SECIS element in the 3'UTR, as revealed by the SECISearch program. In conclusion, our results show, for the first time, the existence of an ascidian iodothyronine outer-ring deiodinase. This raises the hypothesis that, in protochordates, the prohormone T(4) is activated by enzymatic outer-ring deiodination to T(3).

Substitution of cysteine for selenocysteine in the catalytic center of type III iodothyronine deiodinase reduces catalytic efficiency and alters substrate preference.

Human type III iodothyronine deiodinase (D3) catalyzes the conversion of T(4) to rT(3) and of T(3) to 3, 3'-diiodothyronine (T2) by inner-ring deiodination. Like types I and II iodothyronine deiodinases, D3 protein contains selenocysteine (SeC) in the highly conserved core catalytic center at amino acid position 144. To evaluate the contribution of SeC144 to the catalytic properties of D3 enzyme, we generated mutants in which cysteine (D3Cys) or alanine (D3Ala) replaces SeC144 (D3wt). COS cells were transfected with expression vectors encoding D3wt, D3Cys, or D3Ala protein. Kinetic analysis was performed on homogenates with dithiothreitol as reducing cofactor. The Michaelis constant of T(3) was 5-fold higher for D3Cys than for D3wt protein. In contrast, the Michaelis constant of T(4) increased 100-fold. The D3Ala protein was enzymatically inactive. Semiquantitative immunoblotting of homogenates with a D3 antiserum revealed that about 50-fold higher amounts of D3Cys and D3Ala protein are expressed relative to D3wt protein. The relative substrate turnover number of D3Cys is 2-fold reduced for T(3) and 6-fold reduced for T(4) deiodination, compared with D3wt enzyme. Studies in intact COS cells expressing D3wt or D3Cys showed that the D3Cys enzyme is also active under in situ conditions. In conclusion, the SeC residue in the catalytic center of D3 is essential for efficient inner-ring deiodination of T(3) and in particular T(4) at physiological substrate concentrations.

Substitution of cysteine for a conserved alanine residue in the catalytic center of type II iodothyronine deiodinase alters interaction with reducing cofactor.

UNLABELLED: Human type II iodothyronine deiodinase (D2) catalyzes the activation of T(4) to T(3). The D2 enzyme, like the type I (D1) and type III (D3) deiodinases, contains a selenocysteine (SeC) residue (residue 133 in D2) in the highly conserved catalytic center. Remarkably, all of the D2 proteins cloned so far have an alanine two residue-amino terminal to the SeC, whereas all D1 and D3 proteins contain a cysteine at this position. A cysteine residue in the catalytic center could assist in enzymatic action by providing a nucleophilic sulfide or by participating in redox reactions with a cofactor or enzyme residues. We have investigated whether D2 mutants with a cysteine (A131C) or serine (A131S) two-residue amino terminal to the SeC are enzymatically active and have characterized these mutants with regard to substrate affinity, reducing cofactor interaction and inhibitor profile. COS cells were transfected with expression vectors encoding wild-type (wt) D2, D2 A131C, or D2 A131S proteins. Kinetic analysis was performed on homogenates with dithiothreitol (DTT) as reducing cofactor. The D2 A131C and A131S mutants displayed similar Michaelis-Menten constant values for T(4) (5 nM) and reverse T(3) (9 nM) as the wt D2 enzyme. The limiting Michaelis-Menten constant for DTT of the D2 A131C enzyme was 3-fold lower than that of the wt D2 enzyme. The wt and mutant D2 enzymes are essentially insensitive to propylthiouracil [concentration inhibiting 50% of activity (IC(50)) > 2 mM] in the presence of 20 mM DTT, but when tested in the presence of 0.2 mM DTT the IC(50) value for propylthiouracil is reduced to about 0.1 mM. During incubations of intact COS cells expressing wt D2, D2 A131C, or D2 A131S, addition of increasing amounts of unlabeled T(4) resulted in the saturation of [(125)I]T(4) deiodination, as reflected in a decrease of [(125)I]T(3) release into the medium. Saturation first appeared at medium T(4) concentrations between 1 and 10 nM. IN CONCLUSION: substitution of cysteine for a conserved alanine residue in the catalytic center of the D2 protein does not inactivate the enzyme in vitro and in situ, but rather improves the interaction with the reducing cofactor DTT in vitro.