Abstract
Objective
Thyroid hormone (TH) transport represents a critical first step in governing intracellular TH regulation. It is still unknown whether the full repertoire of TH transporters has been identified. Members of the solute carrier (SLC) 22 family have substrates in common with the known TH transporters of the organic anion-transporting peptide family. Therefore, we screened the SLC22 family for TH transporters
Methods
Uptake of 1 nM of iodothyronines or sulfated iodothyronines in COS1 cells expressing SLC22 proteins was performed.
Results
We first tested 25 mouse (m) SLC22 proteins for TH uptake and found that the majority of the organic anion transporter (OAT) clade were capable of 3,3’,5-triiodothyronine and/or thyroxine (T4) transport. Based on phylogenetic tree analysis of the mouse and human (h) SLC22 family, we selected eight hSLC22s that grouped with the newly identified mouse TH transporters. Of these, four tested positive for uptake of one or more substrates, particularly hSLC22A11 showed robust (3-fold over control) uptake of T4. Uptake of sulfated iodothyronines was strongly (up to 17-fold) induced by some SLC22s, most notably SLC22A8, hSLC22A9, mSLC22A27 and mSLC22A29. Finally, the zebrafish orthologues of SLC22A6/8 drOatx and drSlc22a6l also transported almost all (sulfated) iodothyronines tested. The OAT inhibitors lesinurad and probenecid inhibited most SLC22 proteins.
Conclusions
Our results demonstrated that members of the OAT clade of the SLC22 family constitute a novel, evolutionary conserved group of transporters for (sulfated) iodothyronines. Future studies should reveal the relevance of these transporters in TH homeostasis and physiology.
Introduction
Thyroid hormone (TH) is important for development, metabolism and tissue homeostasis. The intracellular bioavailability of TH is dependent on plasma membrane transporters that mediate TH influx and efflux (1). The relevance of TH transporters is clearly demonstrated by the severe disease features in patients with defective monocarboxylate transporter 8 (MCT8) (2). At present, approximately 15 transporters that are able to transport TH have been identified. They comprise MCT8 and MCT10, solute carrier (SLC)17A4 and members of the large amino acid transporters (LATs) and the organic anion-transporting polypeptides (OATPs), as well as the ATP-binding cassette (ABC) family transporter ABCB1 (reviewed in (3)).
It is unknown whether the full repertoire of TH transporters has been identified so far. In this context, the SLC22 family is of potential interest. The SLC22 family is categorized into two major clades based on their substrate preferences: the organic anion transporter (OAT) clade and organic cation transporter (OCT) clade (4). Some members in the OAT major clade share common substrates with the known TH transporters in the OATP family, such as estrone 3 sulfate, prostaglandin E2, estradiol glucuronide and taurocholic acid (5, 6, 7, 8, 9, 10, 11, 12). In addition, the recently identified TH transporter SLC17A4 transports urate (13, 14), which is also a physiologically relevant substrate for some members of the SLC22 family (15, 16, 17, 18, 19).
Apart from iodothyronines, some TH transporters, notably OATP1B1 (20), can also transport iodothyronines that are conjugated at the phenolic group with sulfate. Sulfation of iodothyronines contributes to TH clearance as it greatly enhances inner ring deiodination by the type I deiodinase (DIO1) in the liver and kidney (21). In addition, sulfated TH may be a local source in critical tissues, such as the brain and liver, during fetal development (22). As (sulfated) iodothyronines are organic anions, we hypothesized that some members in the SLC22 family could transport TH and/or its metabolites.
To test our hypothesis, we screened all mouse SLC22 family members and several human SLC22 family members from the OAT clade for the uptake of iodothyronines as well as iodothyronine sulfates. We here report the identification of ten novel iodothyronine (sulfate) transporters, thereby greatly expanding the repertoire of potentially relevant TH transporters.
Materials and methods
Reagents
Nonradioactive 3,3’-diiodothyronine (3,3’-T2), 3,3’,5’-triiodothyronine (rT3), 3,3’,5-triiodothyronine (T3) and thyroxine (T4) were purchased from Sigma-Aldrich (Zwijndrecht, the Netherlands). [125I]-3,3’-T2, [125I]-rT3, [125I]-T3, [125I]-T4, non-radioactive and [125I]-labeled T3 sulfate (T3S) and T4 sulfate (T4S) were prepared as previously described (23). Probenecid and lesinurad (Sigma-Aldrich) were dissolved in 0.15N NaOH and DMSO, respectively.
Constructs, cell culture, transfection and uptake assays
All constructs were commercially purchased or generated from previous studies except for human SLC22A11 (24, 20, 25, 26) (Supplementary Tables 1, 2, 3 and 4 in Supplementary Material 1, see section on supplementary materials given at the end of this article). Generation of the expression construct of human SLC22A11 is described in Supplementary Material 1. Cell culture, transfection and uptake assays were essentially performed as described previously (26, 27). COS1 cells were used for transfection as they have little deiodinase activity that would compromise the transport of the iodothyronines (24). For the uptake assays, we co-transfected the iodothyronine-binding protein CRYM (μ-crystallin) to minimize the efflux of the (sulfated) iodothyronines (26, 28). Full details are provided in Supplementary Material 1.
Phylogenetic tree of mouse and human SLC22 family
The protein sequences of human and mouse SLC22s (accession numbers in Supplementary Table 5 in Supplementary Material 1) were collected from the National Center for Biotechnology Information (NCBI) protein database (https://www.ncbi.nlm.nih.gov/). The phylogenetic tree was generated through http://www.phylogeny.fr/alacarte.cgi using T-coffee multiple alignments of the protein sequences without any curation and constructed with ProtDist/FastDist+BioNJ distances.
Analysis of common genetic variation in newly identified SLC22 TH transporters
We analyzed whether common genetic variation in the SLC22 genes (hSLC22A8, hSLC22A9, hSLC22A11 and hSLC22A24) was associated with serum circulating TH concentrations (free T4 (FT4), and T3/T4 ratios). FT4 analyses were performed using data from 72,167 individuals of the ThyroidOmics Consortium (13, 14). For the FT3/FT4 ratio, we conducted a separate analysis within the two cohorts (SHIP-START-0 and SHIP-TREND-0) of the German population-based Study of Health in Pomerania (29). For the total T3 (TT3)/total T4 (TT4) ratio, a separate analysis was conducted in the Dutch population-based study NIMA (non-invasive measurements of atherosclerosis) (30). Detailed information on the analysis is provided in Supplementary Material 1. All subjects from the above cohorts gave informed consent. The Medical Ethics Committee of the Radboud University Medical Centre approved the study protocol of the NBS/NIMA study and the Medical Ethics Committee of the University of Greifswald approved the study protocol of the SHIP study. Both are in accordance with the Declaration of Helsinki.
Statistical analysis
The statistical analysis was performed using GraphPad Prism version 8.4.0. The sample size and the methods of statistical analysis used were indicated in the figure legends. P < 0.05 was considered significant.
Results
Uptake of iodothyronines by mouse SLC22s
We initially screened all members of the mouse (m) SLC22 family for iodothyronine transport. We first verified whether the mSLC22 proteins were properly expressed at the plasma membrane by immunoblotting biotinylated cell surface proteins with anti-FLAG antibodies. Except for mSLC22A22, whose expression was hardly detectable, all mSLC22s were expressed on the cell membrane (Supplementary Fig. 1 in Supplementary Material 1). The majority of the mSLC22 proteins displayed more than one band suggesting post-translational modifications, di- or oligomerization or protein aggregation, or some protein degradation.
Next, we tested 3,3’-T2, rT3, T3 and T4 uptake in COS1 cells transfected with mSlc22 expression constructs together with the intracellular (sulfated) iodothyronine binding protein CRYM (28, 31). Uptake assays were performed in DPBS/0.1% glucose in the absence (representing a non-stringent screening strategy) or presence (representing a more physiological condition) of 0.1% bovine serum albumin (BSA).
None of the mSLC22 transporters that belong to the OCT major clade (mSLC22A1-5, mSLC22A15, mSLC22A16 and mSLC22A21) (4) facilitated uptake of any of the substrates (Fig. 1 and Supplementary Fig. 2).
Uptake of 1 nM 3,3’-T2, rT3, T3 and T4 in COS1 cells transfected with mouse Slc22s and hCRYM (µ-crystallin). COS1 cells were incubated with the substrates for 10 min (3,3’-T2 and rT3) or 30 min (T3 and T4) in DPBS/0.1% glucose/0.1% BSA. The uptake of mouse Slc22s transfected cells is expressed as fold induction of that in the cells transfected with empty vector (EV). Human LAT1 (for 3,3’-T2 and rT3 uptake) and MCT8 (for T3 and T4 uptake) were used as positive controls (gray bars). Data are represented as mean ± s.d. (n = 3). One-way ANOVA plus Dunnett’s post hoc test (compared to the EV group) was used for statistical analysis. *0.01 < P < 0.05; **0.001 < P ≤ 0.01; ***0.0001 < P ≤ 0.001; ****P ≤ 0.0001.
Citation: European Thyroid Journal 12, 4; 10.1530/ETJ-23-0023
In contrast, several members of the OAT major clade induced the uptake of one or more of the substrates tested. Mouse SLC22A8 induced uptake of all tested substrates, especially 1 nM rT3 and T4 (>2 fold compared to empty vector (EV) transfected cells) (Fig. 1). The closely related OAT members mSLC22A27-30 increased uptake of 1 nM T3 and T4 to a similar extent (Fig. 1). In addition, mSLC22A19 and mSLC22A26 induced T4 uptake but to a lesser extent and only in the absence of BSA (Supplementary Fig. 2). Uptake of 3,3’-T2 by mSLC22A8 was only induced in the presence of BSA. For some transporters, intracellular accumulation of 3,3’-T2 was reduced (Fig. 1 and Supplementary Fig. 2). This may indicate increased efflux of substrates, however, this was not further tested.
Taken together, these results suggest that mSLC22A8 and mSLC22A27-30 transport several iodothyronines, whereas under less stringent conditions, mSLC22A19 and mSLC22A26 transport T4.
Phylogenetic tree of mouse and human SLC22s
Next, we generated a phylogenetic tree to identify the human orthologues of the mSLC22 members that were tested positive for TH uptake. The phylogenetic tree shows that six subclades (OAT, OAT-like, OAT-related, OCT, OCTN and OCT/OCTN related) are separately clustered, in agreement with previous findings (4) and all members that transport iodothyronines reside within the OAT subclade (Fig. 2). Within the OAT clade, six closely related mouse iodothyronine transporters namely mSLC22A27-30, and mSLC22A19 and mSLC22A26, have no human orthologues, but group closely to human (h)SLC22A9, hSLC22A25, hSLC22A10 and hSLC22A24. In addition to these four human SLC22s, we also selected hSLC22A6, hSLC22A8 and hSLC22A11-12 that are grouped in the same cluster to test iodothyronine transport (Fig. 2).
The phylogenetic tree of mouse and human SLC22s. According to literature (reviewed in ‘The SLC22 Transporter Family: A Paradigm for the Impact of Drug Transporters on Metabolic Pathways, Signaling, and Disease’ by Nigam et al. in 2018), the mouse and human SLC22s in gray belong to the organic cation (OCT) major clade and the ones in black belong to the organic anion (OAT) major clade. The OCT major clade consists of three subclades (OCT (SLC22A1-3), OCTN (SLC22A4-5 and 22A21 and OCT/OCTN-related (SLC22A15-16)). The OAT major clade consists of three subclades (OAT-like (SLC22A13-14), OAT-related (SLC22A17-18, 22A23 and 22A31) and OAT (the others)). The mouse SLC22s that are able to transport iodothyronines are underlined in black. The clusters that contain mouse SLC22s transporting iodothyronines were grouped in a black box and the human SLC22 orthologues in the box with arrowheads were selected for further test of uptake of iodothyronines. The number of amino acid substitutions per site was depicted as the length of branches. The line and the number ‘1’ underneath the tree indicate the scale bar of branch length which is one amino acid substitution per site.
Citation: European Thyroid Journal 12, 4; 10.1530/ETJ-23-0023
Uptake of iodothyronines by human SLC22s
The selected hSLC22s showed membrane expression, although expression of hSLC22A25 was relatively low (Supplementary Fig. 3). Like the mouse orthologue, hSLC22A8 induced uptake of all iodothyronines tested (Fig. 3 and Supplementary Fig. 4). Human SLC22A11 induced robust uptake (2.5-fold) of 1 nM T4 but not of any of the other iodothyronines (Fig. 3 and Supplementary Fig. 4). In addition, hSLC22A9 induced weak uptake of T3 and T4 (Fig. 3 and Supplementary Fig. 4). In contrast, hSLC22A6, hSLC22A10, hSLC22A12 and hSLC22A25 did not induce uptake of any of the iodothyronines tested. These results indicate that several of the hSLC22 members of the OAT clade can transport T3 and/or T4.
Uptake of 1 nM 3,3’-T2, rT3, T3 and T4 in COS1 cells transfected with human SLC22s and hCRYM. COS1 cells were incubated with the substrates for 10 min (3,3’-T2 and rT3) or 30 min (T3 and T4) in DPBS/0.1% glucose/0.1% BSA. The uptake of human SLC22s transfected cells is expressed as fold induction of that in the cells transfected with EV. Human LAT1 (for 3, 3’-T2 and rT3 uptake) and MCT8 (for T3 and T4 uptake) were used as positive controls (gray bars). Data are represented as mean ± s.d. (n = 3). One-way ANOVA plus Dunnett’s post hoc test (compared to the EV group) was used for statistical analysis. *0.01 < P < 0.05; ***0.0001 <P ≤ 0.001; ****P ≤ 0.0001.
Citation: European Thyroid Journal 12, 4; 10.1530/ETJ-23-0023
Uptake of sulfated TH by mouse and human SLC22s
Because some OATPs can transport iodothyronine sulfates, we also tested uptake of the sulfated iodothyronines T3S and T4S by mouse and human SLC22 proteins (Fig. 4 and Supplementary Fig. 5). Mouse SLC22A8, mSLC22A27 and mSLC22A29 induced robust uptake (>4-fold for T3S and >3-fold for T4S compared to the EV), with mSLC22A27 showing ~3-fold higher uptake of T4S than the positive control hOATP1B1 (20) (Fig. 4). Of the human SLC22s, hSLC22A8 potently transported T3S and T4S, as evidenced by the ~10-fold increased uptake of T3S and 3-fold increased uptake of T4S, comparable to hOATP1B1 (Fig. 4). In addition, hSLC22A9 efficiently transported T3S (~4-fold) and to a lesser extent T4S (1.8-fold) (Fig. 4). In summary, our results show that some SLC22 members efficiently transport sulfated iodothyronines.
Uptake of 1 nM T3S and T4S in COS1 cells transfected with mouse (A and B) and human (C and D) SLC22s in combination with hCRYM. COS1 cells were incubated with 1 nM T3S (A and C) or T4S (B and D) for 30 min in DPBS/0.1% glucose/0.1% BSA. The uptake of SLC22s transfected cells is expressed as fold induction of that in the cells transfected with EV. Human OATP1B1 was used as a positive control (gray bars). Data are represented as mean ± s.d. (n = 3). One-way ANOVA plus Dunnett’s post hoc test (compared to the EV group) was used for statistical analysis. *0.01< P < 0.05; **0.001 < P ≤ 0.01; ***0.0001 < P ≤ 0.001; ****P ≤ 0.0001.
Citation: European Thyroid Journal 12, 4; 10.1530/ETJ-23-0023
The specificity and efficacy of lesinurad and probenecid for the mouse and human SLC22s
SLC22A8 (also known as OAT3) and SLC22A11 (OAT4), as well as SLC22A12 (URAT1) are important for urate resorption in the kidney (7, 18, 32). Excess serum urate (hyperuricaemia) can cause gout, a form of inflammatory arthritis. Lesinurad and probenecid are FDA-approved drugs for the treatment of gout that act by reducing urate resorption. To determine whether these inhibitors can inhibit (sulfated) iodothyronine uptake by the newly identified TH transporters in the SLC22 family, we tested uptake of the preferred (sulfated) iodothyronines by these transporters in the presence or absence of lesinurad or probenecid (Table 1).
Overview of the newly identified transporters of iodothyronines and/or iodothyronine sulfates in mouse and human SLC22 family. For the specificity and efficacy of lesinurad and probenecid, IC50 values were listed. SLC22A9, SLC22A11 and SLC22A24 are human specific, whereas SLC22A19, SLC22A26-30 are mouse specific.
| Mouse | Human | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Transporter | Substrates | Expression in tissuesa | Substrate tested for inhibition | Lesinurad IC50 | Probenecid IC50 | Substrates | Expression in tissuesb | Substrate tested for inhibition | Lesinurad IC50 | Probenecid IC50 |
| SLC22A8 | 3,3'-T2, rT3, T3, T4, T3S, T4S | Kidney, frontal lobe, brain (endothelial cells)c | T4 | 1.3 μM | 6.0 μM | 3,3'-T2, rT3, T3, T4, T3S, T4S | Kidney, choroid plexus | T3S | 0.6 μM | 3.5 μM |
| SLC22A9 | Not in mouse | T3, T4, T3S, T4S | Liver | T3S | 50 μM | 413 μM | ||||
| SLC22A11 | Not in mouse | T4, T3S | Placenta, kidney, epididymis | T4 | 8.8 μM | 57 μM | ||||
| SLC22A19 | T4 | Kidney | T4 | 5.4 μM | 773 μM | Not in human | ||||
| SLC22A24 | Not in mouse | T4 | Kidney | T3Sd | 11 μM | 24 μM | ||||
| SLC22A26 | rT3, T4, T4S | Liver | T4 | 17 μM | x | Not in human | ||||
| SLC22A27 | 3, 3'-T2, rT3, T3, T4, T3S, T4S | Liver | T4 | x | x | Not in human | ||||
| SLC22A28 | T3, T4 | Liver, kidney | T4 | 26 μM | >>1000 μM | Not in human | ||||
| SLC22A29 | rT3, T3, T4, T3S, T4S | Kidney | T4 | x | x | Not in human | ||||
| SLC22A30 | T3, T4 | Liver | T4 | x | x | Not in human | ||||
aThe tissue expression in mouse was adopted from the mouse ENCODE transcriptome data project (‘A comparative encyclopedia of DNA elements in the mouse genome’ by Yue et al. in 2014) (34). Expression less than five reads per kilo base per million mapped reads (RPKM) are neglected; bThe tissue expression in human was adopted from the HPA datasets from https://www.proteinatlas.org/. Expression less than three transcripts per million (TPM) are neglected; cCell-specific expression in brain was adopted from http://www.brainrnaseq.org/; dT3S was used as the substrate to test for inhibition effect because hSLC22A24 induced T3S uptake more robustly though the induction did not reach statistical significance.
>>1000 μM means within the tested doses, IC50 was not reached; N.T., not tested; X, no inhibition effect.
Lesinurad reduced substrate uptake by all the SLC22 proteins tested in a concentration-dependent manner, except for mSLC22A27, mSLC22A29 and mSLC22A30 (Supplementary Fig. 6). Probenecid inhibited mSLC22A8, hSLC22A8, hSLC22A11 and hSLC22A24 in a concentration-dependent manner but not mSLC22A26-27 and mSLC22A29-30 and only at the highest concentration inhibited mSLC22A19, mSLC22A28 and hSLC22A9 (Supplementary Fig. 7). For mouse and human SLC22A8, the IC50s of lesinurad and probenecid were in the low micromolar range (Table 1). Taken together, lesinurad and probenecid inhibit all the hSLC22s tested and mSLC22A8 (Table 1).
Uptake of (sulfated) iodothyronines for zebrafish SLC22A6 and SLC22A8 orthologues
Among the human (sulfated) iodothyronine transporters, SLC22A9, SLC22A11 and SLC22A24 have only orthologues in mammals but not in other vertebrates. According to the conserved synteny analysis of human and zebrafish (Danio rerio, dr) SLC22/slc22 genes, droatx and drslc22a6l are orthologues of SLC22A6/8 (33). To determine whether (sulfated) iodothyronine transport by OATs is a conserved trait, we tested drOatx and drSlc22a6l for uptake.
Our results showed that both drOatx and drSlc22a6l transported all the tested substrates, except for T3S and T4S transport by drSlc22a6l (Fig. 5). In particular, drOatx is a very potent transporter of 3,3’-T2, rT3, T3S and T4S, comparable with the positive controls hLAT1 and hOATP1B1 (Fig. 5).
Uptake of 1 nM 3,3’-T2, rT3, T3, T4, T3S and T4S in COS1 cells transfected with zebrafish (dr), mouse (mm) and human (hs) SLC22A6 and 22A8 orthologues in combination with hCRYM. COS1 cells were incubated with the substrates for 30 min (10 min for 3,3’-T2 and rT3) in DPBS/0.1% glucose/0.1% BSA. The uptake of SLC22s transfected cells is expressed as fold induction of that in the cells transfected with EV. Human LAT1 (for 3,3’-T2 and rT3), MCT8 (for T3 and T4) and OATP1B1(for T3S and T4S) were used as positive controls (gray bars). Data are represented as mean ± s.d. (n = 3). One-way ANOVA plus Dunnett’s post hoc test (compared to the EV group) was used for statistical analysis. *0.01 < P < 0.05; **0.001 < P ≤ 0.01; ***0.0001 < P ≤ 0.001; ****P ≤ 0.0001.
Citation: European Thyroid Journal 12, 4; 10.1530/ETJ-23-0023
Common genetic variation in newly identified SLC22 TH transporters
As a first step to explore the possible relevance of these newly identified SLC22 transporters for human TH homeostasis, we analyzed whether genetic variations in these SLC22 genes (hSLC22A8, hSLC22A9, hSLC22A11 and hSLC22A24) were associated with serum circulating TH concentrations (FT4, ratios of free T3 (FT3)/FT4 and TT3/TT4. However, none of the single nucleotide polymorphisms (SNPs) that are located in the vicinity of the SLC22 genes are significantly associated after correction for multiple testing (false discovery rate, FDR < 0.05) with the serum FT4 levels, FT3/FT4 or TT3/TT4 ratios (Supplementary Material 2).
Discussion
In the present study, we identified ten novel (sulfated) iodothyronine transporters in the SLC22 family including six mouse-specific and three human-specific members, thereby greatly expanding the repertoire of TH transporters. Among them, seven transporters transport (sulfated) iodothyronines in the more physiological condition of added TH-binding proteins. In addition, we demonstrated that zebrafish orthologues of SLC22A6 and SLC22A8 are capable of transporting almost all the (sulfated) iodothyronines tested, indicating an evolutionary conserved function for (sulfated) iodothyronine transport within the OAT clade. These findings may contribute to the understanding of TH homeostasis in tissues in humans. Furthermore, the identification of (sulfated) iodothyronine transport of mouse and zebrafish homologues paves the way to study the physiological relevance of these transporters in animal models.
Our phylogenetic analysis showed that all newly identified transporters are grouped within the OAT major clade (mainly in the OAT subclade) but not in the OCT major clade (4). In the human genome, most SLC22 genes encoding OATs are located in clusters on chromosome 11, with the exception of SLC22A7. Of these, SLC22A6 and SLC22A8 have phylogenetic and syntenic relationships with the droatx and drSlc22a6l genes, which are clustered on chromosome 21 in the zebrafish genome (33). The transporters encoded by the zebrafish genes both tested positive for nearly all substrates tested, hinting at an evolutionary conserved capacity for (sulfated) iodothyronine transport. This root function is further substantiated by the fact that similar to the zebrafish orthologues, both mouse and human SLC22 had the broadest range of substrates. Gene duplication events resulted in clusters of SLC22 genes some of which specific for either mouse (mSlc22a19 and mSlc22a26-30) or human (SLC22A9, SLC22A11 and SLC22A24). Most of the transporters encoded by these genes retained the capacity for (sulfated) iodothyronine transport, although with some changes in substrate preference. A notable exception is the cluster formed by SLC22A11 and SLC22A12 in the human genome. We found that the human-specific SLC22A11 is a potent T4 transporter, whereas neither hSLC22A12 or mSLC22A12 transported any of the substrates tested.
The physiological function of the newly identified TH transporters for TH homeostasis is as yet unclear. As SLC22 proteins transport a broad range of substrates and have a redundant expression in multiple tissues (Table 1), it is uncertain whether all contribute to TH homeostasis in vivo, particularly those transporters that only showed modest uptake in our in vitro assays. Of interest, many SLC22 proteins are expressed in the liver and kidney (Table 1). Both tissues are important for TH clearance via TH sulfation by sulfotransferases and subsequent deiodination by DIO1. The inner ring deiodinating activity of DIO1 is markedly increased when the substrates are sulfated (21). The balance of influx and efflux of sulfated TH by transporter proteins is therefore an important determinant of the rate of TH clearance and thus circulating TH concentrations. We found several SLC22 members with robust uptake of sulfated iodothyronines, comparable to (hSLC22A8 for T3S and mSLC22A29 for T4S) or even exceeding (mSLC22A27 for T4S) the uptake by hOATP1B1, which is an efficient hepatic transporter for sulfated iodothyronines (20). Human SLC22A8 and SLC22A9 are strongly expressed in human kidney and liver, respectively, whereas the mouse mSlc22A27 and mSlc22A29 are expressed in both tissues (Table 1) (https://www.proteinatlas.org/) (34). We speculate that these transporters may play a role in renal and hepatic clearance of TH via the transport of sulfated iodothyronines. A recent GWAS study showed that the SNPs rs12282281 in the SLC22A9 gene and rs11822642 in the intergenic region between SLC22A9 and HRASLS5 are associated with total serum T4 concentrations and the T3/T4 ratio respectively in a Croatian cohort (35). However, we found no association of SLC22A9 or any other selected SLC22 gene with either FT4 or free or total T3/T4 ratios in the look-up GWAS analysis in the ThyroidOmics Consortium which has a much larger sample size. Of course, our finding does not rule out the population-specific relevance of SLC22A9 and TH levels in the Croatian population. On the other hand, no association of a TH transporter with circulating TH levels does not necessarily mean it has no physiological relevance. For example, common genetic variation in MCT8, mutations which cause neurocognitive phenotypes, has no association with thyroid stimulating hormone (TSH) or FT4 levels (14).
A second process for which TH transport by SLC22 proteins is potentially relevant is the trans-placental transport of TH. Maternal-to-fetal transfer of TH is important for fetal development, particularly during the first trimester when the fetal thyroid gland has not developed yet (36). Although several transporters have been identified, they do not account for all transport, particularly T4 (27). SLC22A11 is strongly expressed in placenta, with its protein present at the basolateral membrane of the syncytiotrophoblasts (37) in the third trimester and its RNA in both the cytotrophoblasts and the syncytiotrophoblasts in the first trimester of pregnancy (38). With its expression in the placenta being higher than MCT8, MCT10, LAT2 and OATPs (39), it may play a role in trans-placental T4 transport to or from the fetal circulation, which is faced by the basolateral surface of the placental syncytium, is a likely possibility. Interestingly, SLC22A11 is specific for humans and to our knowledge no functional homologues from the SLC22 family are expressed in the murine placenta, indicating that putative placental transport of T4 by an SLC22 transporter may be human specific.
Apart from placenta, SLC22A11 is also highly expressed in the kidney. Both SLC22A11 and SLC22A12 are located on the apical membrane of the renal proximal tubule cells, facilitating the resorption of urate (40, 41, 7). Reduced urate secretion from the kidney can cause gout, a chronic disease of monosodium urate crystal deposition in soft tissues due to hyperuricaemia (42). Lesinurad and probenecid are registered drugs against gout that act by blocking urate transport by SLC22A11 and SLC22A12 (43). We found that lesinurad and probenecid also inhibited T4 transport by SLC22A11 with IC50 values in a comparable range as reported for urate (40, 43). In fact, lesinurad and probenecid inhibited (sulfated) iodothyronines by most SLC22 transporters, in particular, hSLC22A8 when tested for T3S transport. Considering the potential role of SLC22 transporters in hepatic and renal TH uptake and metabolism, these drugs could potentially interfere with TH homeostasis in these tissues.
We acknowledge some limitations in our study. First, the transport assays were performed in the presence of CRYM, an intracellular (sulfated) iodothyronine binding protein to minimize the efflux of substrates. This allows us to measure the full extent of substrate uptake, but not efflux capacity. Second, there were differences in the amount of cell surface expression for the different SLC22 proteins. For example, the abundance of mSLC22A22 and hSLC22A25 was particularly low compared to the other transporters and we therefore may have missed the capacity of these proteins to transport (sulfated) iodothyronines. In addition, the differences in expression warrant caution when comparing the capacity to transport (sulfated) iodothyronines between transporters.
In summary, we identified an evolutionary conserved function for several members within the OAT clade of the SLC22 family as (sulfated) iodothyronine transporters. These findings have substantially expanded the repertoire of TH transporters and may contribute to understanding the complexity of TH availability and homeostasis in vivo.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/ETJ-23-0023.
Declaration of interest
The authors declare that they have no conflicts of interest with the contents of this article.
Funding
This study is funded by the EU Horizon 2020 program, ATHENA project, grant number 825161, which is gratefully acknowledged. This publication reflects only the authors’ view, and the European Commission is not responsible for any use that may be made of the information it contains. SHIP is part of the Community Medicine Research net of the University of Greifswald, Germany, which is funded by the Federal Ministry of Education and Research (grants no. 01ZZ9603, 01ZZ0103, and 01ZZ0403), the Ministry of Cultural Affairs as well as the Social Ministry of the Federal State of Mecklenburg-West Pomerania, and the network ‘Greifswald Approach to Individualized Medicine (GANI_MED)’ funded by the Federal Ministry of Education and Research (grant 03IS2061A). Genome-wide data have been supported by the Federal Ministry of Education and Research (grant no. 03ZIK012) and a joint grant from Siemens Healthineers, Erlangen, Germany and the Federal State of Mecklenburg- West Pomerania. The University of Greifswald is a member of the Caché Campus program of the InterSystems GmbH. The Nijmegen Biomedical Study is a population-based survey conducted at the Department for Health Evidence and the Department of Laboratory Medicine of the Radboud University Medical Center. Principal investigators of the Nijmegen Biomedical Study are L.A.L.M. Kiemeney, A.L.M. Verbeek, D.W. Swinkels, and B. Franke.
Author contribution statement
Z.C., M.E.M, W.E.V and R.P.P designed the study. Z.C., W.F., L.J.d.R. and S.Y performed the experiments and processed the experimental data. Z.C. analyzed the data. A.T. and R.B.T.M.S. performed genetic analyses. Z.C., M.E.M, W.E.V, A.T., R.B.T.M.S. and R.P.P wrote the manuscript. All authors critically reviewed, revised and approved the manuscript.
Acknowledgements
The authors thank Selmar Leeuwenburgh for synthesis of the radio-labeled iodothyronines and Ramona E. A. van Heerebeek for her technical assistance in the synthesis of thyroid hormone sulfates. This work was presented at the 43rd Annual Meeting of the European Thyroid Association in 2021 and the meeting abstract was published (DOI: 10.1159/000517526).
References
- 1↑
Friesema EC, Jansen J, & Visser TJ. Thyroid hormone transporters. Biochemical Society Transactions 2005 33 228–232. (https://doi.org/10.1042/BST0330228)
- 2↑
Friesema EC, Grueters A, Biebermann H, Krude H, von Moers A, Reeser M, Barrett TG, Mancilla EE, Svensson J, Kester MH, et al.Association between mutations in a thyroid hormone transporter and severe X-linked psychomotor retardation. Lancet 2004 364 1435–1437. (https://doi.org/10.1016/S0140-6736(0417226-7)
- 3↑
Groeneweg S, van Geest FS, Peeters RP, Heuer H, & Visser WE. Thyroid hormone transporters. Endocrine Reviews 2020 41. (https://doi.org/10.1210/endrev/bnz008)
- 4↑
Nigam SK. The SLC22 transporter family: A paradigm for the impact of drug transporters on metabolic pathways, signaling, and disease. Annual Review of Pharmacology and Toxicology 2018 58 663–687. (https://doi.org/10.1146/annurev-pharmtox-010617-052713)
- 5↑
Kimura H, Takeda M, Narikawa S, Enomoto A, Ichida K, & Endou H. Human organic anion transporters and human organic cation transporters mediate renal transport of prostaglandins. Journal of Pharmacology and Experimental Therapeutics 2002 301 293–298. (https://doi.org/10.1124/jpet.301.1.293)
- 6↑
Kaler G, Truong DM, Sweeney DE, Logan DW, Nagle M, Wu W, Eraly SA, & Nigam SK. Olfactory mucosa-expressed organic anion transporter, Oat6, manifests high affinity interactions with odorant organic anions. Biochemical and Biophysical Research Communications 2006 351 872–876. (https://doi.org/10.1016/j.bbrc.2006.10.136)
- 7↑
Hagos Y, Stein D, Ugele B, Burckhardt G, & Bahn A. Human renal organic anion transporter 4 operates as an asymmetric urate transporter. Journal of the American Society of Nephrology 2007 18 430–439. (https://doi.org/10.1681/ASN.2006040415)
- 8↑
Shin HJ, Anzai N, Enomoto A, He X, Kim DK, Endou H, & Kanai Y. Novel liver-specific organic anion transporter OAT7 that operates the exchange of sulfate conjugates for short chain fatty acid butyrate. Hepatology 2007 45 1046–1055. (https://doi.org/10.1002/hep.21596)
- 9↑
Duan P, & You G. Novobiocin is a potent inhibitor for human organic anion transporters. Drug Metabolism and Disposition: the Biological Fate of Chemicals 2009 37 1203–1210. (https://doi.org/10.1124/dmd.109.026880)
- 10↑
Shiraya K, Hirata T, Hatano R, Nagamori S, Wiriyasermkul P, Jutabha P, Matsubara M, Muto S, Tanaka H, Asano S, et al.A novel transporter of SLC22 family specifically transports prostaglandins and co-localizes with 15-hydroxyprostaglandin dehydrogenase in renal proximal tubules. Journal of Biological Chemistry 2010 285 22141–22151. (https://doi.org/10.1074/jbc.M109.084426)
- 11↑
Roth M, Obaidat A, & Hagenbuch B. OATPs, OATs and OCTs: the organic anion and cation transporters of the SLCO and SLC22A gene superfamilies. British Journal of Pharmacology 2012 165 1260–1287. (https://doi.org/10.1111/j.1476-5381.2011.01724.x)
- 12↑
Yee SW, Stecula A, Chien HC, Zou L, Feofanova EV, van Borselen M, Cheung KWK, Yousri NA, Suhre K, Kinchen JM, et al.Unraveling the functional role of the orphan solute carrier, SLC22A24 in the transport of steroid conjugates through metabolomic and genome-wide association studies. PLOS Genetics 2019 15 e1008208. (https://doi.org/10.1371/journal.pgen.1008208)
- 13↑
Togawa N, Miyaji T, Izawa S, Omote H, & Moriyama Y. A Na+-phosphate cotransporter homologue (SLC17A4 protein) is an intestinal organic anion exporter. American Journal of Physiology. Cell Physiology 2012 302 C1652–C1660. (https://doi.org/10.1152/ajpcell.00015.2012)
- 14↑
Teumer A, Chaker L, Groeneweg S, Li Y, Di Munno C, Barbieri C, Schultheiss UT, Traglia M, Ahluwalia TS, Akiyama M, et al.Genome-wide analyses identify a role for SLC17A4 and AADAT in thyroid hormone regulation. Nature Communications 2018 9 4455. (https://doi.org/10.1038/s41467-018-06356-1)
- 15↑
Kim DK, Kanai Y, Matsuo H, Kim JY, Chairoungdua A, Kobayashi Y, Enomoto A, Cha SH, Goya T, & Endou H. The human T-type amino acid transporter-1: characterization, gene organization, and chromosomal location. Genomics 2002 79 95–103. (https://doi.org/10.1006/geno.2001.6678)
- 16↑
Rizwan AN, & Burckhardt G. Organic anion transporters of the SLC22 family: biopharmaceutical, physiological, and pathological roles. Pharmaceutical Research 2007 24 450–470. (https://doi.org/10.1007/s11095-006-9181-4)
- 17↑
Bahn A, Hagos Y, Reuter S, Balen D, Brzica H, Krick W, Burckhardt BC, Sabolic I, & Burckhardt G. Identification of a new urate and high affinity nicotinate transporter, hOAT10 (SLC22A13). Journal of Biological Chemistry 2008 283 16332–16341. (https://doi.org/10.1074/jbc.M800737200)
- 18↑
Sato M, Iwanaga T, Mamada H, Ogihara T, Yabuuchi H, Maeda T, & Tamai I. Involvement of uric acid transporters in alteration of serum uric acid level by angiotensin II receptor blockers. Pharmaceutical Research 2008 25 639–646. (https://doi.org/10.1007/s11095-007-9401-6)
- 19↑
Sato M, Mamada H, Anzai N, Shirasaka Y, Nakanishi T, & Tamai I. Renal secretion of uric acid by organic anion transporter 2 (OAT2/SLC22A7) in human. Biological and Pharmaceutical Bulletin 2010 33 498–503. (https://doi.org/10.1248/bpb.33.498)
- 20↑
van der Deure WM, Friesema EC, de Jong FJ, de Rijke YB, de Jong FH, Uitterlinden AG, Breteler MM, Peeters RP, & Visser TJ. Organic anion transporter 1B1: an important factor in hepatic thyroid hormone and estrogen transport and metabolism. Endocrinology 2008 149 4695–4701. (https://doi.org/10.1210/en.2008-0169)
- 21↑
Visser TJ. Role of sulfation in thyroid hormone metabolism. Chemico-Biological Interactions 1994 92 293–303. (https://doi.org/10.1016/0009-2797(9490071-x)
- 22↑
Santini F, Chopra IJ, Wu SY, Solomon DH, & Chua GN. Metabolism of 3,5,3'-triiodothyronine sulfate by tissues of the fetal rat: a consideration of the role of desulfation of 3,5,3'-triiodothyronine sulfate as a source of T3. Pediatric Research 1992 31 541–544. (https://doi.org/10.1203/00006450-199206000-00001)
- 23↑
Mol JA, & Visser TJ. Synthesis and some properties of sulfate esters and sulfamates of iodothyronines. Endocrinology 1985 117 1–7. (https://doi.org/10.1210/endo-117-1-1)
- 24↑
Friesema EC, Kuiper GG, Jansen J, Visser TJ, & Kester MH. Thyroid hormone transport by the human monocarboxylate transporter 8 and its rate-limiting role in intracellular metabolism. Molecular Endocrinology 2006 20 2761–2772. (https://doi.org/10.1210/me.2005-0256)
- 25↑
Zevenbergen C, Meima ME, Lima de Souza EC, Peeters RP, Kinne A, Krause G, Visser WE, & Visser TJ. Transport of iodothyronines by human L-type amino acid transporters. Endocrinology 2015 156 4345–4355. (https://doi.org/10.1210/en.2015-1140)
- 26↑
Groeneweg S, Kersseboom S, van den Berge A, Dolcetta-Capuzzo A, van Geest FS, van Heerebeek REA, Arjona FJ, Meima ME, Peeters RP, Visser WE, et al.In vitro characterization of human, mouse, and zebrafish MCT8 orthologues. Thyroid 2019 29 1499–1510. (https://doi.org/10.1089/thy.2019.0009)
- 27↑
Chen Z, van der Sman ASE, Groeneweg S, de Rooij LJ, Visser WE, Peeters RP, & Meima ME. Thyroid hormone transporters in a human placental cell model. Thyroid 2022 32 1129–1137. (https://doi.org/10.1089/thy.2021.0503)
- 28↑
Visser WE, Wong WS, van Mullem AA, Friesema EC, Geyer J, & Visser TJ. Study of the transport of thyroid hormone by transporters of the SLC10 family. Molecular and Cellular Endocrinology 2010 315 138–145. (https://doi.org/10.1016/j.mce.2009.08.003)
- 29↑
Völzke H, Schössow J, Schmidt CO, Jürgens C, Richter A, Werner A, Werner N, Radke D, Teumer A, Ittermann T, et al.Cohort profile update: the study of health in Pomerania (SHIP). International Journal of Epidemiology 2022 51 e372–e383. (https://doi.org/10.1093/ije/dyac034)
- 30↑
Galesloot TE, Vermeulen SH, Swinkels DW, de Vegt F, Franke B, den Heijer M, de Graaf J, Verbeek ALM, & Kiemeney LALM. Cohort profile: the Nijmegen Biomedical Study (NBS). International Journal of Epidemiology 2017 46 1099–1100j. (https://doi.org/10.1093/ije/dyw268)
- 31↑
Groeneweg S, van Geest FS, Chen Z, Farina S, van Heerebeek REA, Meima ME, Peeters RP, Heuer H, Medici M, & Visser WE. Functional characterization of the novel and specific thyroid hormone transporter SLC17A4. Thyroid 2022 32 326–335. (https://doi.org/10.1089/thy.2021.0257)
- 32↑
Sun HL, Wu YW, Bian HG, Yang H, Wang H, Meng XM, & Jin J. Function of uric acid transporters and their inhibitors in hyperuricaemia. Frontiers in Pharmacology 2021 12 667753. (https://doi.org/10.3389/fphar.2021.667753)
- 33↑
Mihaljevic I, Popovic M, Zaja R, & Smital T. Phylogenetic, syntenic, and tissue expression analysis of slc22 genes in zebrafish (Danio rerio). BMC Genomics 2016 17 626. (https://doi.org/10.1186/s12864-016-2981-y)
- 34↑
Yue F, Cheng Y, Breschi A, Vierstra J, Wu W, Ryba T, Sandstrom R, Ma Z, Davis C, Pope BD, et al.A comparative encyclopedia of DNA elements in the mouse genome. Nature 2014 515 355–364. (https://doi.org/10.1038/nature13992)
- 35↑
Gunjaca I, Matana A, Boutin T, Torlak V, Punda A, Polasek O, Boraska V, Hayward C, Zemunik T, & Barbalic M. Genome-wide association meta-analysis for total thyroid hormone levels in Croatian population. Journal of Human Genetics 2019 64 473–480. (https://doi.org/10.1038/s10038-019-0586-4)
- 36↑
Patel J, Landers K, Li H, Mortimer RH, & Richard K. Delivery of maternal thyroid hormones to the fetus. Trends in Endocrinology and Metabolism 2011 22 164–170. (https://doi.org/10.1016/j.tem.2011.02.002)
- 37↑
Ugele B, St-Pierre MV, Pihusch M, Bahn A, & Hantschmann P. Characterization and identification of steroid sulfate transporters of human placenta. American Journal of Physiology. Endocrinology and Metabolism 2003 284 E390–E398. (https://doi.org/10.1152/ajpendo.00257.2002)
- 38↑
Vento-Tormo R, Efremova M, Botting RA, Turco MY, Vento-Tormo M, Meyer KB, Park JE, Stephenson E, Polanski K, Goncalves A, et al.Single-cell reconstruction of the early maternal-fetal interface in humans. Nature 2018 563 3 47–3 53. (https://doi.org/10.1038/s41586-018-0698-6)
- 39↑
Saben J, Zhong Y, McKelvey S, Dajani NK, Andres A, Badger TM, Gomez-Acevedo H, & Shankar K. A comprehensive analysis of the human placenta transcriptome. Placenta 2014 35 125–131. (https://doi.org/10.1016/j.placenta.2013.11.007)
- 40↑
Enomoto A, Kimura H, Chairoungdua A, Shigeta Y, Jutabha P, Cha SH, Hosoyamada M, Takeda M, Sekine T, Igarashi T, et al.Molecular identification of a renal urate anion exchanger that regulates blood urate levels. Nature 2002 417 447–452. (https://doi.org/10.1038/nature742)
- 41↑
Motohashi H, Sakurai Y, Saito H, Masuda S, Urakami Y, Goto M, Fukatsu A, Ogawa O, & Inui KI. Gene expression levels and immunolocalization of organic ion transporters in the human kidney. Journal of the American Society of Nephrology 2002 13 866–874. (https://doi.org/10.1681/ASN.V134866)
- 42↑
Dalbeth N, Merriman TR, & Stamp LK. Gout. Lancet 2016 388 2039–2052. (https://doi.org/10.1016/S0140-6736(1600346-9)
- 43↑
Miner JN, Tan PK, Hyndman D, Liu S, Iverson C, Nanavati P, Hagerty DT, Manhard K, Shen Z, Girardet JL, et al.Lesinurad, a novel, oral compound for gout, acts to decrease serum uric acid through inhibition of urate transporters in the kidney. Arthritis Research and Therapy 2016 18 214. (https://doi.org/10.1186/s13075-016-1107-x)
