Abstract
Mutations in the gene encoding the thyroid hormone transporter, monocarboxylate transporter 8 (MCT8), underlie severe mental retardation. We wanted to understand the functional consequences of a series of missense mutations in MCT8 in order to identify therapeutic options for affected patients. We established cell lines stably expressing 12 MCT8 variants in JEG1 and MDCK1 cells. The cell lines were characterized according to MCT8 mRNA and protein expression, tri-iodothyronine (T3) transport activity, substrate KM characteristics, surface expression, and responsiveness to T3 preincubation and chemical chaperones. Functional activities of ins235V and L568P MCT8 mutants depend on the cell type in which they are expressed. These mutants and R271H exhibited considerable transport activity when present at the cell surface as verified by surface biotinylation and kinetic analysis. Most mutants, however, were inactive in T3 transport even when present at the cell surface (e.g. S194F, A224V, ΔF230, L512P). Preincubation of G558D with T3 increased T3 uptake in MDCK1 cells to a small, but significant, extent. Chemical chaperones were ineffective. The finding that the cell type determines surface expression and T3 transport activities of missense mutants in MCT8 may be important to understand phenotypic variability among carriers of different mutations. In particular, the clinical observation that the severity of derangements of thyroid hormone levels does not correlate with mental impairments of the patients may be based on different residual activity of mutant MCT8 in different cell types.
Introduction
Mutations affecting the gene encoding monocarboxylate transporter 8 (MCT8; SLC16A2) have been identified in patients suffering from a syndrome associated with X-linked mental retardation and with elevated serum tri-iodothyronine (T3; Dumitrescu et al. 2004, Friesema et al. 2004, Schwartz et al. 2005), which was formerly clinically described as the Allan–Herndon–Dudley syndrome (AHDS).
MCT8 is a 12 membrane-pass sodium- and ATP-independent transport protein which has been identified earlier as an active T3 transport protein (Friesema et al. 2003). Other members of the MCT gene family, e.g. MCT1 and MCT10, have been shown to transport lactate or amino acids respectively, and MCT10 also transports T3 (Halestrap & Meredith 2004, Friesema et al. 2008). Thyroid hormones have long been considered as hydrophobic molecules able to efficiently cross the plasma membrane, but mounting evidence suggests that T3 and thyroxine (T4) need specific transport proteins for membrane crossing (Hennemann et al. 2001, Visser et al. 2007). Congenital hypothyroidism may lead to severe mental retardation if left untreated. Neonatal screening programs allow us to identify and treat these patients early, and most affected children attain normal intelligence (Grüters et al. 2003). In the brain, thyroid hormone is required for the timely migration of neurons, formation of synaptic contacts, and myelination (Zoeller et al. 2002).
Since MCT8 is expressed, among other tissues, in neurons and pituitary cells (Heuer et al. 2005), it has been expected that mutations in MCT8 may deprive developing neurons of adequate T3 signalling leading to mental impairment, albeit, the phenotypes of MCT8-deficient patients are not compatible with untreated congenital hypothyroidism (Dumitrescu et al. 2004, Friesema et al. 2004, Holden et al. 2005, Kakinuma et al. 2005, Schwartz et al. 2005, Maranduba et al. 2006, Herzovich et al. 2007, Jansen et al. 2007). In addition, the apparent lack of feedback inhibition of TSH expression in the pituitary suggests that pituitary thyrotroph cells are not able to correctly sense circulating T3 levels. In contrast, decreased serum cholesterol and elevated sex hormone binding globulin (SHBG) levels in affected patients point to hepatic hyperthyroidism (Dumitrescu et al. 2004, Friesema et al. 2004).
Transgenic mice have been engineered in which the murine Mct8 gene was disrupted (Dumitrescu et al. 2006, Trajkovic et al. 2007). These mice exhibit greatly increased serum T3 in the face of low/normal T4 and normal TSH levels (Dumitrescu et al. 2006, Trajkovic et al. 2007). Like the patients, they exhibit correspondingly decreased serum cholesterol. Brain uptake of T3, but not T4, is completely inhibited in Mct8−/y mice. In line with this observation, expression and activity of Dio2 is increased and Dio3 is decreased. However, surprisingly no neurological phenotype was observed in these mice (Dumitrescu et al. 2006, Trajkovic et al. 2007), possibly because of neuronal expression of additional T3 transporters in the mouse (Wirth et al. 2009).
Over 20 mutations in MCT8 have been found in patients so far. Patients are usually identified in their first year of life with severe muscular hypotonia. Most patients never develop speech or independent walking. It was noted that there is some phenotypic variation among patients with different mutations (Schwartz et al. 2005, Friesema et al. 2006, Frints et al. 2008, Jansen et al. 2008) – and there is even some variability among the phenotypes of patients within the same family (Frints et al. 2008). Phenotypic variation may point to residual T3 transport activity of mutated MCT8 depending on the type of mutation. This theoretically opens the possibility to aim therapeutically at an increase of the activity of mutated MCT8. Potential treatment strategies for patients with large genomic deletions or frameshift mutations would require gene therapy in the nervous system and are therefore not established. In contrast, since it has been suggested that some MCT8 mutations prevent the protein from reaching the plasma membrane (Jansen et al. 2008), the application of chemical or pharmacological chaperones may increase plasma membrane trafficking of mutated MCT8 (Bernier et al. 2004). In order to study the molecular defects of individual mutant MCT8 proteins and their response towards pharmacological treatments in vitro, we have established stable cell lines expressing a series of 12 missense MCT8 mutations found in AHDS patients and analyzed their protein expression, T3-uptake activity, and cell surface exposure. We found that most of the missense mutants were readily expressed in several cell types. We then observed that a number of these mutants are also able to transport T3, but that this activity was related to their surface expression, which – as we found – depends considerably on the cell type studied. Kinetic analyses of partially active MCT8 mutants showed that some mutations primarily affect membrane exposure (e.g. L568P), while others more directly impinge on T3 transport (e.g. ins235V). These findings may bear significance for future treatment strategies, since the results allow distinguish missense mutations in MCT8 according to their molecular pathomechanisms.
Materials and methods
Cloning, site-directed mutagenesis, and transient expression
Mutations were introduced into human (amino acids 1–613) N-terminally HA-tagged MCT8 cDNA by overlap extension PCR using the primers hMCT8-HindIII-HA-fwd: 5′-AAGCTTGACATGTACCCATACGACGTCCCAGACTACGCTATGGGGAGAGGAGGAG-3′ and genrev 5′-TTAGATTGGTTCCTCAGGGTTGG-3′ with primers introducing the mutations as listed in Supplementary Table 1, see supplementary data in the online version of the Journal of Molecular Endocrinology at http://jme.endocrinology-journals.org/content/vol43/issue6/. In the primer sequences, the start and stop codons are printed in bold face and the HA-tag is denoted in italics. The PCR fragments were subcloned into pGEM-Teasy (Promega), sequenced from both sides, and then cloned into pcDNA3.1 (Invitrogen) using the restriction enzymes HindIII and XbaI. Expression of full-length MCT8 was verified by transient transfection into cultured cells followed by immunoblotting with an antiserum made in rabbit and directed against a recombinant N-terminal peptide of human MCT8 (amino acids 52–155; ATLAS, Stockholm, Sweden) or the HA-tag (not shown). Stability of variant MCT8 protein was tested by incubating freshly harvested membrane fraction protein lysates at 37 °C for up to 3 h with and without the addition of a protease inhibitor cocktail (Complete Mini, Roche) followed by immunoblotting (Supplementary Figure 1, see supplementary data in the online version of the Journal of Molecular Endocrinology at http://jme.endocrinology-journals.org/content/vol43/issue6/).
Cell culture, transfection, and generation of stable cell lines
Cell lines were cultured in a humidified atmosphere at 37 °C and 5% CO2. The media used were DMEM-F12 (1:1), 5% FCS and Pen/Strep (only JEG1 and MDCK1) for HEK-293, HeLa, JEG1 and MDCK1 cells respectively. JEG1 and MDCK1 cells were selected for low background T3 uptake activity (not shown) and lack of endogenous MCT8 expression as judged by western and northern blotting (Fig. 1 and Supplementary Figure 2, see supplementary data in the online version of the Journal of Molecular Endocrinology at http://jme.endocrinology-journals.org/content/vol43/issue6/). Cells at 50% confluency were transfected with expression plasmids encoding wild-type (WT), mutants, and empty vector and exposed to selective pressure with 200 μg G418/ml. After 2 weeks, colonies derived from single clones were picked and subcultured. Three to six clones of each variant in both cell types were expanded in six well plates, protein harvested, dot blots performed all in parallel for each cell type and probed with an anti-MCT8 antibody. Three clones each expressing levels of MCT8 similar to WT were selected and stored in liquid nitrogen. One clone for each variant was randomly chosen from the three frozen, G418 withdrawn, continued MCT8 expression ascertained by SDS-PAGE and immunoblotting, and used for the following experiments.
Expression of transiently transfected MCT8 variants in mammalian cell lines. (A) Cell lines were transfected with human MCT8 (1–613) and MCT8 (75–613) cDNA expression vectors and expression of MCT8 protein was determined by immunoblotting. Initiation at Met75 occurs also in cells transfected with full length (1–613) MCT8 depending on cell type. ‘+’ and ‘−’ indicate the MCT8 construct transfected. Molecular mass markers are indicated on the right. (B) Transient transfection into HeLa cells of MCT8 (amino acids 1–613) variants found in human patients. All missense mutants except L512P were readily detectable as full length MCT8 protein. WT, wild-type MCT8. (C) Stable transfection into JEG1 cells of MCT8 (amino acids 1–613) variants found in human patients. All missense mutants including L512P were readily detectable as full length MCT8 protein. (D) Stable transfection into canine MDCK1 cells of MCT8 (amino acids 1–613) variants found in human patients. All missense mutants except L512P were readily detectable as full length MCT8 protein.
Citation: Journal of Molecular Endocrinology 43, 6; 10.1677/JME-09-0043
Expression of transiently transfected MCT8 variants in mammalian cell lines. (A) Cell lines were transfected with human MCT8 (1–613) and MCT8 (75–613) cDNA expression vectors and expression of MCT8 protein was determined by immunoblotting. Initiation at Met75 occurs also in cells transfected with full length (1–613) MCT8 depending on cell type. ‘+’ and ‘−’ indicate the MCT8 construct transfected. Molecular mass markers are indicated on the right. (B) Transient transfection into HeLa cells of MCT8 (amino acids 1–613) variants found in human patients. All missense mutants except L512P were readily detectable as full length MCT8 protein. WT, wild-type MCT8. (C) Stable transfection into JEG1 cells of MCT8 (amino acids 1–613) variants found in human patients. All missense mutants including L512P were readily detectable as full length MCT8 protein. (D) Stable transfection into canine MDCK1 cells of MCT8 (amino acids 1–613) variants found in human patients. All missense mutants except L512P were readily detectable as full length MCT8 protein.
Citation: Journal of Molecular Endocrinology 43, 6; 10.1677/JME-09-0043
Expression of transiently transfected MCT8 variants in mammalian cell lines. (A) Cell lines were transfected with human MCT8 (1–613) and MCT8 (75–613) cDNA expression vectors and expression of MCT8 protein was determined by immunoblotting. Initiation at Met75 occurs also in cells transfected with full length (1–613) MCT8 depending on cell type. ‘+’ and ‘−’ indicate the MCT8 construct transfected. Molecular mass markers are indicated on the right. (B) Transient transfection into HeLa cells of MCT8 (amino acids 1–613) variants found in human patients. All missense mutants except L512P were readily detectable as full length MCT8 protein. WT, wild-type MCT8. (C) Stable transfection into JEG1 cells of MCT8 (amino acids 1–613) variants found in human patients. All missense mutants including L512P were readily detectable as full length MCT8 protein. (D) Stable transfection into canine MDCK1 cells of MCT8 (amino acids 1–613) variants found in human patients. All missense mutants except L512P were readily detectable as full length MCT8 protein.
Citation: Journal of Molecular Endocrinology 43, 6; 10.1677/JME-09-0043
Northern blotting
For northern blot analysis, total RNA was isolated from stably transfected JEG1 and MDCK1 cells using peqGOLD TriFast (PeqLab, Erlangen, Germany). For electrophoresis 15 μg of total RNA were separated using a 2% agarose gel with 4.3% formaldehyde followed by blotting onto a nylon membrane (Schleicher & Schuell, Dassel, Germany). 32P-labeled RNA probe was generated by labeling a 1.3 kb BamHI/XbaI cDNA probe cut from hMCT8-pcDNA3.1 according to the manufacturer's protocol (NEBlot Kit; New England Biolabs, Frankfurt, Germany).
T3 uptake assay and chaperone use
One day before the experiments, cells were trypsinized, counted, 100 000 cells were set aside for western blotting against MCT8, and 200 000 cells per well were seeded into 12-well plates. 125I–T3 (GE Healthcare, Munich, Germany; 11.8 MBq/ml, 150 μCi/μg) was purified from iodide by reverse phase chromatography and eluted in EtOH:NH3 (49:1) which was removed by a gentle stream of nitrogen gas. On the day of the experiment, the cell culture medium was replaced by DMEM F12 (1:1) without serum. The cells were exposed to 10 nM of 125I–T3 in DMEM:F12 for 3 (MDCK1 cells) or 15 min (JEG1 cells as determined in pilot studies), and the medium removed. After a single wash with PBS, the cells were lysed in 40 mM NaOH, and cell associated radioactivity was quantified with a gamma counter (Wizard; Perkin Elmer, Rodgau, Germany). Each experiment was performed on at least three different days with different batches of cells in triplicate. In order to pool the data, radioactivity associated with empty vector-transfected clones was subtracted as background and the activity associated with the WT MCT8 clone was defined as 100%. Specific T3-uptake activity was calculated by dividing the % uptake by relative protein expression on the plasma membrane (mean of two to three experiments, see below).
For determination of apparent KM values of WT and mutant MCT8, MCDK1 cells stably expressing WT, ins235V, R271H, and L568P were selected and incubated for 3 min with 125I–T3 at concentrations ranging from 500 nM to 12 μM. A Michaelis-Menten mechanism was assumed for calculations (Graph Pad 4.0; Graph Pad, La Jolla, CA, USA).
In order to test the effect of chemical chaperones, the following substances were added to the cells 24 h before performing uptake assay: 80 mM trimethylamine-N-oxide (TMAO, Sigma), 50 mM glycerol (Sigma), 2 mM tauryl-ursodeoxycholic acid (TUDCA, Sigma) or 2 mM 4-phenyl-butyric acid (4-PBA, Tocris, BIOZOL, Eching, Germany). In order to test whether T3 can serve as a pharmacological chaperone, cells were pre-incubated with 10 nM T3 for 30 min before medium exchange and subsequent T3 uptake measurements.
Surface biotinylation and western blotting
Surface biotinylation was performed on cells grown in 75 cm2 flasks using the Pierce Cell Surface Protein Isolation Kit according to the manufacturer's instructions with minor modifications as follows. We found that half the amount of cells recommended are still sufficient to detect biotinylated MCT8 and thus used only half volumes of reagents where appropriate. After elution from the avidin resin with dithiothreitol (DTT), we precipitated the biotinylated protein with acetone, removed excess DTT by washing (in order to reduce interference with the Bradford protein assay) and determined the protein content of the eluate. Equal amounts of 5 μg of biotinylated protein (or 80 μg of total cellular protein fraction) were then applied to 10% SDS-PAGE gels, separated, electroblotted, and probed with the MCT8 antibody. Relative surface translocation was assessed by densitometry of X-ray films (ImageJ; NIH, Bethesda, MD, USA) and expressed relative to WT MCT8. Only full length MCT8 was considered, excluding degradation products. Equal protein loading on all lanes of the membranes was ascertained by reversible Amido black B staining. Every experiment in each cell type was performed at least twice with similar results. Equal loading of lanes was further tested by probing with β-actin antibody.
Results
Efficient expression of MCT8 variants in several cell lines
The open reading frames of primate MCT8 genes are longer than in most other mammalian species so far analyzed. Met75 in human MCT8 is identical with Met1 in rodent, cat, and dog MCT8. Therefore, there is uncertainty regarding the human translational start codon in vivo. We thus transiently transfected long (amino acids 1–613) and short (amino acids 75–613) variants of WT human MCT8 cDNA into four different human and canine cell lines. Interestingly, all cell types were able to initiate translation at Met75 from the vector encoding (1–613) MCT8, albeit at lower rates (Fig. 1A). We then introduced into full-length (1–613) human MCT8 cDNA a series of 12 missense mutations reflecting the base changes found in patients afflicted with AHDS (OMIM 300523). In contrast to earlier reports (Jansen et al. 2005, 2008), most of the missense mutants were readily detectable by western blotting when transiently expressed in HeLa, JEG1, and MDCK1 cell lines (Fig. 1B and not shown). We then speculated that the stability of the MCT8 mutant proteins may be shortened and therefore we incubated membrane protein fractions from HeLa cells transiently transfected with MCT8 expression constructs at 37 °C for up to 3 h in the presence or absence of protease inhibitors. Most mutants demonstrated almost identical stability as WT MCT8, while L471P and ΔF230 appeared to be slightly less stable in vitro (Supplementary Figure 1).
Cell surface translocation and T3-uptake activity of MCT8 variants
We then established stable cell lines for all MCT8 variants in human chorioncarcinoma JEG1 cells and canine kidney MDCK1 cells (Fig. 1C and D; Supplementary Figure 2). Using these cell lines, we compared 125I–T3 uptake mediated by mutant MCT8 with WT MCT8. In order to express T3-uptake as specific activity relative to MCT8 at the plasma membrane, we performed selective biotinylation of cell surface-exposed proteins and quantified the amount of biotinylated variant MCT8 in relation to WT MCT8. We noticed that specific T3-uptake by mutant MCT8 was different between JEG1 (Fig. 2A) and MDCK1 cells (Fig. 2B). Such differences may rest in part with differential protein expression or stability. For example, the G558D mutant is apparently unstable in JEG1, but not in MDCK and HeLa cells (see also Fig. 1 and Supplementary Figure 1). In addition, while in JEG1 cells only the R271H mutant was active in both cell lines similarly as previously reported (Jansen et al. 2007). In MDCK1 cells, ins235V and L568P, in addition to R271H, were highly active. The difference is likely associated with plasma membrane translocation and protein degradation. For example, L568P efficiently translocates to the plasma membrane in MDCK1, but not JEG1 cells. Similarly, ins235V translocates only moderately to the membrane in JEG1, and is partially degraded. Thus, the disease mechanism for some mutant MCT8 proteins involves cell type-specific deficiency of plasma membrane translocation which secondarily impinges on transport activity.
MCT8 surface translocation and 125I–T3 uptake depends on the cell line. JEG1 (A) and MDCK1 (B) cells were stably transfected with expression vectors encoding MCT8 variants identified in human patients. Whole cellular lysates (top panels) were compared by immunoblotting for MCT8 with the biotinylated and affinity-purified plasma membrane protein fraction (bottom panels). Uptake of cells transfected with empty vector was considered background and subtracted. Wild-type (WT) MCT8 T3 uptake was set to 100% and the data were normalized relative to MCT8 plasma membrane exposure to yield a measure for the specific activity. Data represent mean±s.e.m. of at least three independent T3-uptake experiments performed in triplicate and two biotinylation studies per cell type. *P<0.05, **P<0.01, ***P<0.001 different from WT. n.d., not determined (because of absent plasma membrane expression (G558D, V235M) or erratic replicates (L434W)). #Values negative after background subtraction, but reproducible).
Citation: Journal of Molecular Endocrinology 43, 6; 10.1677/JME-09-0043
MCT8 surface translocation and 125I–T3 uptake depends on the cell line. JEG1 (A) and MDCK1 (B) cells were stably transfected with expression vectors encoding MCT8 variants identified in human patients. Whole cellular lysates (top panels) were compared by immunoblotting for MCT8 with the biotinylated and affinity-purified plasma membrane protein fraction (bottom panels). Uptake of cells transfected with empty vector was considered background and subtracted. Wild-type (WT) MCT8 T3 uptake was set to 100% and the data were normalized relative to MCT8 plasma membrane exposure to yield a measure for the specific activity. Data represent mean±s.e.m. of at least three independent T3-uptake experiments performed in triplicate and two biotinylation studies per cell type. *P<0.05, **P<0.01, ***P<0.001 different from WT. n.d., not determined (because of absent plasma membrane expression (G558D, V235M) or erratic replicates (L434W)). #Values negative after background subtraction, but reproducible).
Citation: Journal of Molecular Endocrinology 43, 6; 10.1677/JME-09-0043
MCT8 surface translocation and 125I–T3 uptake depends on the cell line. JEG1 (A) and MDCK1 (B) cells were stably transfected with expression vectors encoding MCT8 variants identified in human patients. Whole cellular lysates (top panels) were compared by immunoblotting for MCT8 with the biotinylated and affinity-purified plasma membrane protein fraction (bottom panels). Uptake of cells transfected with empty vector was considered background and subtracted. Wild-type (WT) MCT8 T3 uptake was set to 100% and the data were normalized relative to MCT8 plasma membrane exposure to yield a measure for the specific activity. Data represent mean±s.e.m. of at least three independent T3-uptake experiments performed in triplicate and two biotinylation studies per cell type. *P<0.05, **P<0.01, ***P<0.001 different from WT. n.d., not determined (because of absent plasma membrane expression (G558D, V235M) or erratic replicates (L434W)). #Values negative after background subtraction, but reproducible).
Citation: Journal of Molecular Endocrinology 43, 6; 10.1677/JME-09-0043
There are MCT8 variants which are primarily defective in T3 transport, even if they are efficiently translocated to the plasma membrane. For example, membrane expression of ins189I, S194F, A224V, ΔF230, and L512P is apparently possible in both cell lines, although the respective mutants are essentially inactive in T3-uptake.
Biochemical properties of MCT8 variants
In order to better characterize the biochemical properties of MCT8 mutants which retained T3 transport activity, we undertook a kinetic study. The KM value for T3 in uptake assays is an intrinsic feature of the transporter molecule and is independent of the number of molecules exposed to the cell surface. The Vmax, in contrast, depends on the number of molecules involved. In order to adjust for slight quantitative differences in plasma membrane expression, we normalized T3 uptake activity to relative amounts of MCT8 at the plasma membrane as above and determined Vmax from the specific uptake activities in MDCK1 cells (Fig. 3). Both ins235V (
Kinetic analysis of 125I–T3 uptake by mutant MCT8 proteins in MDCK1 cells. (A) Cell-associated radioactivity depending on substrate (T3) concentration. Lines represent fitted binding isotherms. All data points were determined in triplicate and the mean values are plotted. WT (wild-type MCT); ins235V; R271H; L568P; ΔF230 and S194F were also tested, but did not show T3 uptake above background. The experiment was performed two times with similar results. (B) Eadie–Hofstee plot based on data from (A). L568P displays the same KM value as WT. While R271H displays an intermediate KM value, ins235V exhibits the highest KM value. KM values were not approximated from the plot, but were directly calculated from the data using GraphPad software.
Citation: Journal of Molecular Endocrinology 43, 6; 10.1677/JME-09-0043
Kinetic analysis of 125I–T3 uptake by mutant MCT8 proteins in MDCK1 cells. (A) Cell-associated radioactivity depending on substrate (T3) concentration. Lines represent fitted binding isotherms. All data points were determined in triplicate and the mean values are plotted. WT (wild-type MCT); ins235V; R271H; L568P; ΔF230 and S194F were also tested, but did not show T3 uptake above background. The experiment was performed two times with similar results. (B) Eadie–Hofstee plot based on data from (A). L568P displays the same KM value as WT. While R271H displays an intermediate KM value, ins235V exhibits the highest KM value. KM values were not approximated from the plot, but were directly calculated from the data using GraphPad software.
Citation: Journal of Molecular Endocrinology 43, 6; 10.1677/JME-09-0043
Kinetic analysis of 125I–T3 uptake by mutant MCT8 proteins in MDCK1 cells. (A) Cell-associated radioactivity depending on substrate (T3) concentration. Lines represent fitted binding isotherms. All data points were determined in triplicate and the mean values are plotted. WT (wild-type MCT); ins235V; R271H; L568P; ΔF230 and S194F were also tested, but did not show T3 uptake above background. The experiment was performed two times with similar results. (B) Eadie–Hofstee plot based on data from (A). L568P displays the same KM value as WT. While R271H displays an intermediate KM value, ins235V exhibits the highest KM value. KM values were not approximated from the plot, but were directly calculated from the data using GraphPad software.
Citation: Journal of Molecular Endocrinology 43, 6; 10.1677/JME-09-0043
Effects of pharmacological chaperones on T3 transport activity of mutant MCT8
If the stability of the three-dimensional structure of a membrane protein is significantly reduced, it may be retained in the endoplasmic reticulum (ER) by quality control mechanisms or even be degraded. We therefore tested whether chemical chaperones previously shown to enhance surface expression of mutant membrane proteins, are able to increase T3 transport by MCT8 mutants in MDCK1 cells. For this experiment, we selected mutants that retained partial activity and showed surface expression in at least one cell type, and incubated the cells with chemical chaperones for 24 h before the T3 uptake assay. Neither chemical chaperone, 4-PBA, TUDCA, glycerol, nor TMAO did enhance cellular T3 uptake in the selected mutant MCT8 cell lines (Fig. 4A). Some mutant integral membrane proteins adopt a native conformation in the presence of a ligand and thus pass quality control in the ER. Since we knew that MDCK1 cells exhibit low T3 uptake activity in the absence of transfected MCT8, we speculated that T3 may be available to unstable MCT8 mutants retained in the ER. After an initial screening comprising all mutant MCT8 cell lines, we selected G558D and L471P and pre-incubated MDCK1 cells transgenic for these mutants with 10 nM T3 before performing T3 uptake measurements. While no effect was observed for L471P, we observed a small, but signficant and reproducible, increase in T3 uptake for G558D with T3 pre-treatment (Fig. 4B). However, even with T3 pre-incubation, we were not able to determine a meaningful KM value for the G558D mutant.
Effect of pharmacological treatments on T3 uptake mediated by mutant MCT8. (A) Twenty-four hours preincubation with chemical chaperones trimethylaminoxide (TMAO), glycerol, tauroursodeoxycholic acid (TUDCA), or 4-phenylbutyric acid (4-PBA) was not effective in enhancing T3 uptake mediated by mutant MCT8. (B) Thirty minutes preincubation with T3 as pharmacological chaperone encreased T3 uptake by G558D, but not L471P mutant MCT8.
Citation: Journal of Molecular Endocrinology 43, 6; 10.1677/JME-09-0043
Effect of pharmacological treatments on T3 uptake mediated by mutant MCT8. (A) Twenty-four hours preincubation with chemical chaperones trimethylaminoxide (TMAO), glycerol, tauroursodeoxycholic acid (TUDCA), or 4-phenylbutyric acid (4-PBA) was not effective in enhancing T3 uptake mediated by mutant MCT8. (B) Thirty minutes preincubation with T3 as pharmacological chaperone encreased T3 uptake by G558D, but not L471P mutant MCT8.
Citation: Journal of Molecular Endocrinology 43, 6; 10.1677/JME-09-0043
Effect of pharmacological treatments on T3 uptake mediated by mutant MCT8. (A) Twenty-four hours preincubation with chemical chaperones trimethylaminoxide (TMAO), glycerol, tauroursodeoxycholic acid (TUDCA), or 4-phenylbutyric acid (4-PBA) was not effective in enhancing T3 uptake mediated by mutant MCT8. (B) Thirty minutes preincubation with T3 as pharmacological chaperone encreased T3 uptake by G558D, but not L471P mutant MCT8.
Citation: Journal of Molecular Endocrinology 43, 6; 10.1677/JME-09-0043
Discussion
Mutations in the MCT8 gene in humans lead to a severe form of mental and psychomotor retardation. To date, no efficient treatment is available. The observation that patients carrying the S194F, L434W, and L568P mutations may develop rudimentary speech or independent, although awkward, walking (Schwartz et al. 2005) suggests that at least these MCT8 variants retain some residual transport activity in vivo. Consistent with these clinical observations, these mutants retained residual transport activity in JEG3 cells (Jansen et al. 2008). We show here that MCT8 mutants ins235V, R271H and L568P exhibit significant T3 transport activity in JEG1 or MDCK1 cells. We also show that differential activity of the mutants in the two cell lines, depends on surface translocation to the plasma membrane. In contrast to results obtained in JEG3 cells (Jansen et al. 2008), we were not able to detect significant residual activity of S194F and L434W, although at least S194F was efficiently transported to the plasma membrane in both cell lines. This is reminiscent of the report of Jansen et al. in which affinity to bromoacetyl-T3 was high for L568P, but low for the S194F und L434W mutants. The mutants ins235V, R271H, and L568P exhibit normal or close to normal KM values and thus we demonstrated for the first time that MCT8 mutants so far considered inactive may act as functional T3 transporters, if their plasma membrane translocation is successful (Fig. 5). Plasma membrane translocation of multipass membrane proteins is often impaired in genetic diseases affecting integral membrane proteins, e.g. in cystic fibrosis or diabetes insipidus. Therapies are being developed to increase the fraction of (mutant) protein reaching the plasma membrane in vivo (Burrows et al. 2000). Folding of multipass transmembrane proteins is a complicated and still incompletely understood process (von Heijne 2006). Unstable or unsucessfully assembled membrane proteins are identified in the ER and are degraded by cytosolic proteasomes after a process called retrotranslocation – subsequently called the endoplasmic reticulum associated degradation (ERAD) pathway (Kleizen & Braakman 2004). In an attempt to test for possible induction of the unfolded-protein-response by mutant MCT8 proteins, we tested in all stable MDCK1 cell lines for the expression of XBP1s and Chop transcripts by RT-PCR. We found in none of the mutant cell lines increased expression of these transcripts (data not shown) in accordance with another report (James et al. 2008).
Summary of biochemical phenotypes of mutant MCT8. (1) Wild-type MCT8 mediates cellular T3 uptake at the plasma membrane. (2) Some MCT8 mutants display increased KM for T3 uptake, but are partially functional (e.g. R271H). (3) Other mutants may translocate to the plasma membrane, but are entirely deficient in T3 transport (A224V). (4) Mutants like L568P are enzymatically fully active, but depending on the cellular environment, may not translocate to the plasma membrane. (5) Some mutants are incapable of plasma membrane translocation. (6) The G558D mutant is expressed at low levels or subject to degradation, but a small fraction of molecules may reach the plasma membrane and is active as T3 transporter. Incubation with T3 as pharmacological chaperone enhances T3 uptake by G558D (based on results from MDCK1 cells).
Citation: Journal of Molecular Endocrinology 43, 6; 10.1677/JME-09-0043
Summary of biochemical phenotypes of mutant MCT8. (1) Wild-type MCT8 mediates cellular T3 uptake at the plasma membrane. (2) Some MCT8 mutants display increased KM for T3 uptake, but are partially functional (e.g. R271H). (3) Other mutants may translocate to the plasma membrane, but are entirely deficient in T3 transport (A224V). (4) Mutants like L568P are enzymatically fully active, but depending on the cellular environment, may not translocate to the plasma membrane. (5) Some mutants are incapable of plasma membrane translocation. (6) The G558D mutant is expressed at low levels or subject to degradation, but a small fraction of molecules may reach the plasma membrane and is active as T3 transporter. Incubation with T3 as pharmacological chaperone enhances T3 uptake by G558D (based on results from MDCK1 cells).
Citation: Journal of Molecular Endocrinology 43, 6; 10.1677/JME-09-0043
Summary of biochemical phenotypes of mutant MCT8. (1) Wild-type MCT8 mediates cellular T3 uptake at the plasma membrane. (2) Some MCT8 mutants display increased KM for T3 uptake, but are partially functional (e.g. R271H). (3) Other mutants may translocate to the plasma membrane, but are entirely deficient in T3 transport (A224V). (4) Mutants like L568P are enzymatically fully active, but depending on the cellular environment, may not translocate to the plasma membrane. (5) Some mutants are incapable of plasma membrane translocation. (6) The G558D mutant is expressed at low levels or subject to degradation, but a small fraction of molecules may reach the plasma membrane and is active as T3 transporter. Incubation with T3 as pharmacological chaperone enhances T3 uptake by G558D (based on results from MDCK1 cells).
Citation: Journal of Molecular Endocrinology 43, 6; 10.1677/JME-09-0043
Reduction of the dielectric constant of the cellular interior caused by so-called ‘chemical chaperones’ (glycerol, TMAO, TUDCA, 4-PBA) diminishes the thermodynamic penalty for non-perfect protein conformations and allows mutant proteins to evade the ERAD machinery (Schulz et al. 2002, Bernier et al. 2004). Another potential strategy to increase the expression of mutated plasma membrane proteins is to present a membrane permeant ligand for the target protein, a ‘pharmacological chaperone’ that induces its correct three-dimensional folding (Bernier et al. 2004). Such ligands may even represent enzymatic inhibitors, but their tight binding intracellularly provides the extra free energy for stabilization of the target protein. Providing T3 preincubation to mutant MCT8 was the only manipulation effective in significantly enhancing T3 uptake, although only in one mutant, G558D. These findings are disappointing with regard to the lack of therapy for patients suffering from mutations in MCT8. While the search for more effective drugs that may enhance T3 transport by mutant MCT8 should continue, it is equally important to identify those patients who likely do not benefit and who may suffer potential side effects of the therapy without the chance of therapeutic success. According to our data, carriers of e.g. ins189I and A224V mutations are not likely to benefit from any therapy aimed at increasing plasma membrane expression of MCT8, since their MCT8 protein is not deficient in surface translocation and yet is entirely inactive.
Other experimental therapies for MCT8-deficient patients included the attempt to override the apparent resistance to thyroid hormone by further increasing T3 in the patient, but no clinical improvement could be demonstrated (Biebermann et al. 2005). In contrast, Wemeau et al. ameliorated the thyrotoxic state by combined T4/propyl-thio-uracil (PTU) treatment, because they wanted to achieve weight gain in the cachexic patient with the R271H mutation. Although their treatment did not improve the neurological impairment of the patient, significant weight gain was achieved (Wemeau et al. 2008).
One alternative possible treatment option has not been explored in patients to our knowledge. Synthetic ligands for thyroid hormone receptors may be able to cross the blood–brain barrier, and possibly the neuronal plasma membrane, independent of MCT8. Since it has been shown that under conditions of hypothyroidism or due to thyroid hormone receptor mutations unliganded nuclear thyroid hormone receptors exert repressive action on gene expression (Morte et al. 2002), providing to MCT8-deficient patients a synthetic ligand able to reach neuronal nuclei without the aid of MCT8, which may at least alleviate that part of the neurological deficits not directly caused by developmental derangements. Application of such compounds, together with T4/PTU, may restore peripheral euthyroidism and still increase thyroid hormone receptor activity in the brain.
In the report of Frints et al. (2008) a family was described carrying a missense mutation in the first ATG codon of MCT8. While one sibling exhibited a phenotype compatible with AHDS, a brother carrying the same mutation was apparently not affected. It is still not clear whether the inactivation of the first ATG in MCT8 of the patient was the sole reason for his mental retardation, but two observations should be considered: primates may initiate MCT8 translation at the ‘first’ ATG, while rodents, for example, utilize the ATG homologous to Met75 in humans. Initial experiments on T3 transport by Friesema et al. (2003) and Biebermann et al. (2005) used the rat and the (75–613) version of human MCT8 demonstrating significant T3-transport activity of the shorter MCT8 isoform. Thus, we speculate that the healthy sibling, unlike his deceased brother, may be able to initiate MCT8 translation at Met75. In support of this, we showed that, in vitro, several human cell lines produced a significant fraction of (75–613) MCT8 protein along with full length (1–613) MCT8 (compare Figs 1A and D and 2).
Considering the cell type differences in MCT8 translocation to the plasma membrane, demonstrated here for the first time, may help explain why the levels of plasma T3 do not correlate with mental development in MCT8-deficient patients (Friesema et al. 2006). The cellular make-up of protein-folding chaperones or ERAD components likely differs between neurons, pituitary thyrotrophs, and hepatocytes and thus different MCT8 missense mutants may be handled differently in the various cells. For example, if mutant MCT8 partially works in some neurons, but not in the pituitary, T3 will be very high, but mental impairment less severe. In the reverse situation, plasma T3 will be closer to normal, but the mental impairment maximal as in null mutations. Such differences may, however, complicate each attempt to design a pharmacological treatment strategy for MCT8-deficient patients and should be considered in experimental design and the choice of analytical parameters.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
Financial support was provided by Deutsche Forschungsgemeinschaft DFG, project SFB665 TP A7, and EnForCé, Technologiestiftung Berlin.
Acknowledgements
The authors wish to thank Dr Andreas Kaufmann, Charité, for providing JEG1 cells and Dr Petra Ambrugger for initial experiments.
References
Bernier V, Lagace M, Bichet DG & Bouvier M 2004 Pharmacological chaperones: potential treatment for conformational diseases. Trends in Endocrinology and Metabolism 15 222–228.
Biebermann H, Ambrugger P, Tarnow P, von Moers A, Schweizer U & Grueters A 2005 Extended clinical phenotype, endocrine investigations and functional studies of a loss-of-function mutation A150V in the thyroid hormone specific transporter MCT8. European Journal of Endocrinology 153 359–366.
Burrows JA, Willis LK & Perlmutter DH 2000 Chemical chaperones mediate increased secretion of mutant alpha 1-antitrypsin (alpha 1-AT) Z: a potential pharmacological strategy for prevention of liver injury and emphysema in alpha 1-AT deficiency. PNAS 97 1796–1801.
Dumitrescu AM, Liao XH, Best TB, Brockmann K & Refetoff S 2004 A novel syndrome combining thyroid and neurological abnormalities is associated with mutations in a monocarboxylate transporter gene. American Journal of Human Genetics 74 168–175.
Dumitrescu AM, Liao XH, Weiss RE, Millen K & Refetoff S 2006 Tissue-specific thyroid hormone deprivation and excess in monocarboxylate transporter (mct) 8-deficient mice. Endocrinology 147 4036–4043.
Friesema EC, Ganguly S, Abdalla A, Manning Fox JE, Halestrap AP & Visser TJ 2003 Identification of monocarboxylate transporter 8 as a specific thyroid hormone transporter. Journal of Biological Chemistry 278 40128–40135.
Friesema EC, Grueters A, Biebermann H, Krude H, von Moers A, Reeser M, Barrett TG, Mancilla EE, Svensson J & Kester MH et al. 2004 Association between mutations in a thyroid hormone transporter and severe X-linked psychomotor retardation. Lancet 364 1435–1437.
Friesema EC, Jansen J, Heuer H, Trajkovic M, Bauer K & Visser TJ 2006 Mechanisms of disease: psychomotor retardation and high T3 levels caused by mutations in monocarboxylate transporter 8. Nature Clinical Practice. Endocrinology and Metabolism 2 512–523.
Friesema ECH, Jansen J, Jachtenberg JW, Visser WE, Kester MH & Visser TJ 2008 Effective cellular uptake and efflux of thyroid hormone by human monocarboxylate transporter 10. Molecular Endocrinology 22 1357–1369.
Frints SG, Lenzner S, Bauters M, Jensen LR, Van Esch H, des Portes V, Moog U, Macville MV, van Roozendaal K & Schrander-Stumpel CT et al. 2008 MCT8 mutation analysis and identification of the first female with Allan–Herndon–Dudley syndrome due to loss of MCT8 expression. European Journal of Human Genetics 16 1029–1037.
Grüters A, Biebermann H & Krude H 2003 Neonatal thyroid disorders. Hormone Research 59 24–29.
Halestrap AP & Meredith D 2004 The SLC16 gene family-from monocarboxylate transporters (MCTs) to aromatic amino acid transporters and beyond. Pflügers Archiv 447 619–628.
von Heijne G 2006 Membrane-protein topology. Nature Reviews. Molecular Cell Biology 7 909–918.
Hennemann G, Docter R, Friesema EC, de Jong M, Krenning EP & Visser TJ 2001 Plasma membrane transport of thyroid hormones and its role in thyroid hormone metabolism and bioavailability. Endocrine Reviews 22 451–476.
Herzovich V, Vaiani E, Marino R, Dratler G, Lazzati JM, Tilitzky S, Ramirez P, Iorcansky S, Rivarola MA & Belgorosky A 2007 Unexpected peripheral markers of thyroid function in a patient with a novel mutation of the MCT8 thyroid hormone transporter gene. Hormone Research 67 1–6.
Heuer H, Maier MK, Iden S, Mittag J, Friesema EC, Visser TJ & Bauer K 2005 The monocarboxylate transporter 8 linked to human psychomotor retardation is highly expressed in thyroid hormone-sensitive neuron populations. Endocrinology 146 1701–1706.
Holden KR, Zuniga OF, May MM, Su H, Molinero MR, Rogers RC & Schwartz CE 2005 X-linked MCT8 gene mutations: characterization of the pediatric neurologic phenotype. Journal of Child Neurology 20 852–857.
James SR, Franklyn JA, Reaves BJ, Smith VE, Chan SY, Barrett TG, Kilby MD & McCabe CJ 2008 Monocarboxylate transporter 8 (MCT8) in neuronal cell growth. Endocrinology 150 1961–1969.
Jansen J, Friesema EC, Milici C & Visser TJ 2005 Thyroid hormone transporters in health and disease. Thyroid 15 757–768.
Jansen J, Friesema EC, Kester MH, Milici C, Reeser M, Gruters A, Barrett TG, Mancilla EE, Svensson J & Wemeau JL et al. 2007 Functional analysis of monocarboxylate transporter 8 mutations identified in patients with X-linked psychomotor retardation and elevated serum triiodothyronine. Journal of Clinical Endocrinology and Metabolism 92 2378–2381.
Jansen J, Friesema EC, Kester MH, Schwartz CE & Visser TJ 2008 Genotype–phenotype relationship in patients with mutations in thyroid hormone transporter MCT8. Endocrinology 149 2184–2190.
Kakinuma H, Itoh M & Takahashi H 2005 A novel mutation in the monocarboxylate transporter 8 gene in a boy with putamen lesions and low free T4 levels in cerebrospinal fluid. Journal of Pediatrics 147 552–554.
Kleizen B & Braakman I 2004 Protein folding and quality control in the endoplasmic reticulum. Current Opinion in Cell Biology 16 343–349.
Maranduba CM, Friesema EC, Kok F, Kester MH, Jansen J, Sertie AL, Passos-Bueno MR & Visser TJ 2006 Decreased cellular uptake and metabolism in Allan–Herndon–Dudley syndrome (AHDS) due to a novel mutation in the MCT8 thyroid hormone transporter. Journal of Medical Genetics 43 457–460.
Morte B, Manzano J, Scanlan T, Vennstrom B & Bernal J 2002 Deletion of the thyroid hormone receptor alpha 1 prevents the structural alterations of the cerebellum induced by hypothyroidism. PNAS 99 3985–3989.
Schulz A, Sangkuhl K, Lennert T, Wigger M, Price DA, Nuuja A, Gruters A, Schultz G & Schoneberg T 2002 Aminoglycoside pretreatment partially restores the function of truncated V(2) vasopressin receptors found in patients with nephrogenic diabetes insipidus. Journal of Clinical Endocrinology and Metabolism 87 5247–5257.
Schwartz CE, May MM, Carpenter NJ, Rogers RC, Martin J, Bialer MG, Ward J, Sanabria J, Marsa S & Lewis JA et al. 2005 Allan–Herndon–Dudley syndrome and the monocarboxylate transporter 8 (MCT8) gene. American Journal of Human Genetics 77 41–53.
Trajkovic M, Visser TJ, Mittag J, Horn S, Lukas J, Darras VM, Raivich G, Bauer K & Heuer H 2007 Abnormal thyroid hormone metabolism in mice lacking the monocarboxylate transporter 8. Journal of Clinical Investigation 117 627–635.
Visser WE, Friesema EC, Jansen J & Visser TJ 2007 Thyroid hormone transport by monocarboxylate transporters. Best Practice and Research. Clinical Endocrinology and Metabolism 21 223–236.
Wemeau JL, Pigeyre M, Proust-Lemoine E, d'Herbomez M, Gottrand F, Jansen J, Visser TJ & Ladsous M 2008 Beneficial effects of propylthiouracil plus l-thyroxine treatment in a patient with a mutation in MCT8. Journal of Clinical Endocrinology and Metabolism 93 2084–2088.
Wirth EK, Roth S, Blechschmidt C, Hölter SH, Becker L, Racz I, Zimmer A, Klopstock T, Gailus-Durner V & Fuchs H et al. 2009 Neuronal T3 uptake and behavioral phenotype of mice deficient in Mct8, the neuronal T3 transporter mutated in Allan–Herndon–Dudley syndrome. Journal of Neuroscience 29 9439–9449.
Zoeller TR, Dowling AL, Herzig CT, Iannacone EA, Gauger KJ & Bansal R 2002 Thyroid hormone, brain development, and the environment. Environmental Health Perspectives 110 355–361.
*(A Kinne and S Roth contributed equally to the experiments presented in this manuscript)
