Identification and Characterization of 3-Iodothyronamine Intracellular Transport

Abstract

3-Iodothyronamine (T₁AM) is a naturally occurring thyroid hormone metabolite with distinct biological effects that are opposite those of thyroid hormone. The known molecular targets of T₁AM include both plasma membrane and intracellular proteins, suggesting that intracellular transport of T₁AM may be an important component of its action, although no uptake mechanism has yet been described. Using various human cell lines, we show that, indeed, cellular uptake of T₁AM occurs in multiple cell types and that this process involves specific, saturable, and inhibitable transport mechanisms. These mechanisms are sodium and chloride independent, pH dependent, thyronamine specific, and do not involve the likely candidate transporters of other monoamines, organic cations, or thyroid hormones. A large-scale RNA interference screen targeting the entire solute carrier superfamily of transporter genes reveals that the transport of T₁AM into cells involves multiple transporters, and we identify eight transporters that may contribute to the uptake of T₁AM in HeLa cells. This type of transporter small interfering RNA screening approach can be used in general to identify the constellation of transporters that participate in the intracellular disposition of compounds.

A large-scale RNAi screen targeting the entire SLC superfamily of transporter genes reveals that the transport of T₁AM into cells involves multiple transporters, and eight transporters that may contribute to the uptake of T₁AM in HeLa cells are identified.

Thyronamines are a recently discovered class of compounds arising from the decarboxylation of thyroid hormone (1), a classic endocrine hormone that acts by regulating transcription of target genes involved in many important physiological actions, including regulation of growth, development, and metabolic functions (2). One of these thyronamines, 3-iodothyronamine (T₁AM), is a biogenic amine that is found in vertebrate tissues as well as in the circulatory system. Intraperitoneal injection of T₁AM into mice results in profound hypothermia and bradycardia within minutes, a time scale too rapid to be explained by a transcriptional mechanism (1). In addition, T₁AM administration rapidly induces hyperglycemia in mice (3) and rapidly triggers a shift in fuel usage toward lipids and away from carbohydrates in both mice and Siberian hamsters (4). Interestingly, whereas thyroid hormone exerts most of its actions over a period of hours to days, certain rapidly occurring effects of thyroid hormone have been reported but remain unexplained (5,6). The rapid, nontranscriptional effects of T₁AM may be a novel mechanism for regulation of thyroid hormone function in response to constantly changing physiological conditions. Insight into the mechanism of action of this thyroid hormone metabolite would thus greatly contribute to our current understanding of thyroid endocrinology.

Like other trace amines, T₁AM is a potent agonist of the rat and mouse trace amine associated receptor 1 (TAAR1), members of the G protein-coupled receptor family (1). T₁AM may also have a neuromodulatory role as an inhibitor of the dopamine and norepinephrine transporters responsible for the reuptake of these classical neurotransmitters as well as the vesicular monoamine transporter VMAT2, an intracellular transporter that packages monoamines into synaptic vesicles (7). Whereas TAAR1 signaling mechanisms and modulation of monoamine transport may help explain some of the pharmacological effects of thyronamines in vivo, a greater understanding of the actions of T₁AM is needed.

Other structurally related compounds, including the biogenic amine neurotransmitters dopamine, serotonin, and norepinephrine, are translocated across plasma membranes by various transporters (8,9). As such, we hypothesized that there might likewise exist plasma membrane transport mechanisms for the uptake of T₁AM. This transport mechanism could serve to terminate the signal of T₁AM at its extracellular receptors or provide a means of recycling the compound, analogous to the critical function of reuptake transporters of the monoamine neurotransmitters.

Thyroid hormone itself is transported across the cell membrane by a variety of transporters, the dysfunction of which results in certain disease states (10,11,12,13). Despite our knowledge of T₁AM's action at the TAAR1 G protein-coupled receptor and its neuromodulatory activities, the mechanism of physiological action of T₁AM remains largely unknown. Although T₁AM does not bind the nuclear thyroid hormone receptors (1), it may nevertheless have other important roles inside the cell yet to be discovered. An understanding of the cellular transport of T₁AM would provide insight into its mechanisms of action and possible role in regulation of thyroid hormone activity.

With the goal of expanding our knowledge of the molecular mechanisms underlying T₁AM action, the aim of this study was to determine the mechanisms by which T₁AM enters cells. In particular, we were interested in characterizing the processes involved in the intracellular uptake of T₁AM and identifying specific transporters that participate in its intracellular disposition.

Materials and Methods

T₁AM transport assay

Cell lines were grown in the appropriate recommended complete growth medium (American Type Culture Collection, Manassas, VA) for the particular cell line at 37 C with 5% CO₂ and 95% humidity. In preparation for uptake assays, cells were seeded into 24-well tissue culture plates and uptake experiments were performed the following day. Cells were washed and preincubated with prewarmed KRTH [120 mm NaCl, 4.7 mm KCl, 2.2 mm CaCl₂, 1.2 mm MgSO₄, 1.2 mm KH₂PO₄, 5 mm Tris, 10 mm HEPES (pH 7.4)] for 15 min at 37 C. Uptake was initiated by the addition of a tracer amount of ¹²⁵I-T₁AM, synthesized as described previously (14), with or without various concentrations of unlabeled compounds diluted in KRTH. Uptake was terminated after 20 min at 37 C, the cells were washed twice with cold KRTH and solubilized in 1% sodium dodecyl sulfate, and the accumulated radioactivity was determined by scintillation counting. For uptake assays performed in sodium- or chloride-free buffer, KRTH was modified by replacing sodium with choline or chloride with gluconate, respectively. For uptake assays performed at varying pH, HCl or NaOH was added to unmodified KRTH to achieve the desired pH. Transport activity for each condition was measured in triplicate on at least three separate occasions. Data given for relative uptake of T₁AM show representative uptake for a single experiment done in triplicate.

Generation of stable cell lines

Stable cell lines were used for all of the experiments testing the function of individual transporters. FlpIn HEK 293 cells (Invitrogen, Carlsbad, CA) were maintained in DMEM of high glucose supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin (University of California, San Francisco, Cell Culture Facility, San Francisco, CA). Stable cell lines were created by introducing a construct containing the complete coding sequence (CDS) of the particular transporter gene cloned into the pcDNA5/FRT vector or the pcDNA5/FRT vector alone, according to the manual of the FlpIn system (Invitrogen). The monocarboxylate transporter MCT8 cDNA was generously supplied by the laboratory of Theo Visser (Erasmus University Medical Center, Rotterdam, The Netherlands) and used to construct the MCT8 HEK stable cell line. Stably transfected HEK FlpIn cells were selected after 48 h by the addition of 75 μg/ml hygromycin. The plasma membrane monoamine transporter (PMAT) MDCK stable cell line and corresponding pcDNA3 empty vector cell line were received from the laboratory of Joanne Wang (University of Washington, Seattle, WA) (15). For transport assays performed with control substrates, cells were incubated with the appropriate radiolabeled substrate for the particular transporter tested under transport assay conditions identical to those for T₁AM described above.

Small interfering RNA (siRNA) transfection and screening

A custom siRNA library consisting of three unique targets each against 403 transporters, for a total of 1209 siRNAs, was obtained from Ambion (Austin, TX). Positive control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) siRNA and negative control siRNA were also from Ambion. HeLa cells were transfected with individual siRNAs using NeoFX (Ambion) using the standard method of reverse transfection in 96-well tissue culture plates according to the manufacturer's protocols. Forty-eight hours after transfection, cells were washed and preincubated with prewarmed KRTH and T₁AM assays were conducted as described above. T₁AM uptake for transporter siRNA-transfected cells was compared relative to that of negative control siRNA-transfected cells.

For each transfection, a set of cells were also transfected with GAPDH siRNA and GAPDH gene knockdown was verified by quantitative RT-PCR (qRT-PCR) as a positive control for successful transfection. Transporter gene knockdown of the final eight transporter candidates was also ultimately verified by qRT-PCR. Gene expression levels were calculated using the comparative threshold cycle (Ct) method. Forty-eight hours after siRNA transfection, HeLa cells were harvested using the Cells-to-Signal protocol (Ambion), and gene-specific primers and probes were obtained as TaqMan assays (Applied Biosystems, Foster City, CA). The Ct values resulting from amplification were normalized to either phosphoglycerate kinase 1 (PGK1) when assessing GAPDH knockdown or GAPDH when assessing transporter knockdown to give ΔCt values. The target ΔCt values were then normalized with the ΔCt values of the calibrator samples, which consisted of cells transfected with negative control siRNA, to give ΔΔCt values. The formula 2^-ΔΔCt was used to obtain the normalized gene expression levels.

Transport experiments performed for transporters identified by siRNA screening

The eight transporters eventually identified as potential transporters of T₁AM were individually cloned, and the cDNA containing the complete CDS of each transporter was introduced into the pcDNA5/FRT vector. Stable HEK FlpIn cells were then generated as previously mentioned. For some of the transporters with high expression levels in HEK cells, MDCK or CHO FlpIn stable cell lines were also constructed. Two variants of solute carrier (SLC) O3A1 were tested using CHO FlpIn stable cell lines kindly provided by the laboratory of Bruno Stieger (University Hospital Zurich, Zurich, Switzerland) (16). Transport assays were conducted for each stable cell line under experimental conditions identical to those for T₁AM and control substrates described above.

Statistical analyses

Statistical analyses were performed with GraphPad Prism software (version 4.00; GraphPad Inc., San Diego, CA), with values expressed as means ± sd.

Results

Intracellular uptake of T₁AM in multiple cell lines involves facilitated transport mechanisms

To characterize T₁AM uptake in cultured cells, we examined several diverse cell lines for specific uptake of T₁AM by incubating the cells with ¹²⁵I-T₁AM (14) either alone or in the presence of excess unlabeled T₁AM. The variety of cell lines screened included rodent cell lines L6 (rat skeletal muscle) and BC3H1 (mouse brain tumor); insect Sf9 cells (pupal ovarian tissue); and the human cell lines CAKI-1 (kidney), U2OS (bone), Hep G2 (liver), HISM (smooth intestine), HeLa (cervix), HEK 293 (kidney), and 293T (kidney) cells. For all cell lines screened, we observed significantly reduced uptake of ¹²⁵I-T₁AM in the presence of 50 μm unlabeled T₁AM, suggesting the existence of specific transport mechanisms of T₁AM in vitro. Relative uptake of ¹²⁵I-T₁AM was similar among all cell lines tested, and in most cell lines, the addition of 50 μm unlabeled T₁AM resulted in an approximately 3-fold reduction in uptake (Fig. 1A). By varying the concentration of unlabeled T₁AM during the uptake experiments, we observed a dose-dependent inhibition of radiolabeled T₁AM uptake with an IC₅₀ of approximately 7.7 μm in HeLa cells (Fig. 1B). Collectively, the data indicate that T₁AM uptake occurs in multiple cell types and involves facilitated transport mechanisms. Observation of T₁AM uptake in cultured cell lines derived from a variety of tissue sources suggests that T₁AM may have actions throughout the body and is consistent with its endogenous presence in several different vertebrate tissues.

Figure 1.

Identification and properties of T₁AM transport. A, Specific uptake of ¹²⁵I-T₁AM can be demonstrated in a variety of cell lines. B, Dose-dependent inhibition of ¹²⁵I-T₁AM with unlabeled T₁AM in HeLa cells. This response suggests a specific mechanism of T₁AM uptake with an IC₅₀ of about 7.7 μm. C, Effect of thyronamines on T₁AM uptake. Uptake is inhibited to varying degrees and in a dose-dependent manner by all of the other thyronamines, with the exception of T₄AM. D, Effect of sodium and chloride on T₁AM uptake. Experiments performed in uptake buffer with and without sodium and chloride show that uptake is independent of these ions. For sodium-free buffer, sodium was replaced by choline, and for chloride-free buffer, chloride was replaced by gluconate. E, Effect of pH on T₁AM uptake. Top panel, Experiments performed in uptake buffer at different pH show that, whereas total uptake increases with increasing pH, the fold increase in uptake over background remains the same. Bottom panel, Subtraction of background uptake shows an increase in ¹²⁵I-T₁AM uptake with increasing pH, suggesting that T₁AM uptake may be driven by an outwardly directed proton gradient. The increase in total and background T₁AM uptake at higher pH may be a result of an increased fraction of the deprotonated form of the monoamine, which can more readily diffuse across the plasma membrane. In each of the graphs, relative ¹²⁵I-T₁AM uptake values are normalized either to the maximum uptake signal (disintegrations per minute radioactivity counts) obtained with ¹²⁵I-T₁AM incubation alone (A, C, and D) or to the overall maximum uptake signal obtained for all the conditions depicted in the graph (B and E).

Other thyronamines compete with T₁AM for uptake

We next determined the effect of other thyronamines on T₁AM uptake. The complete panel of thyronamines has been chemically synthesized (1) and consists of the nine possible iodination states, including the noniodinated T₀AM. At least two of the thyronamines, T₁AM and T₀AM, are present endogenously (7). Because of the close structural similarity among all the thyronamines, which differ only by the number and position of iodine molecules, it seemed likely that several may be transported by the same mechanism as T₁AM. Because radiolabeled versions of the other thyronamines were not available, we indirectly tested for their uptake by conducting competition experiments with T₁AM.

With the exception of T₄AM, we observed a dose-dependent decrease in T₁AM uptake for all thyronamines, suggesting competition with T₁AM uptake. Whereas the extent of competition was similar among the remaining thyronamines, rT₃AM, T₀AM, and 3′,5′-T₂AM appeared to be slightly less potent (Fig. 1C). Although 3,3′-T₂AM reduced the uptake of ¹²⁵I-T₁AM to a greater extent at 50 μm than T₁AM itself, the difference was not statistically significant and unlabeled T₁AM was among the most potent of the thyronamines at competing with uptake of radiolabeled T₁AM.

Sodium- and chloride-independent, pH-dependent uptake of T₁AM

To further characterize T₁AM transport, uptake experiments were performed in buffer lacking sodium or chloride to determine whether T₁AM uptake was dependent on these ions. Similar levels of uptake were observed for the different buffer compositions, revealing that transport is sodium and chloride independent (Fig. 1D). These results suggest that T₁AM transport does not involve a sodium and chloride cotransport mechanism.

The pH of the uptake buffer was also varied to investigate the effect on T₁AM transport. The fold increase in uptake over background (i.e. in the presence of excess unlabeled T₁AM) remained constant; however, after subtracting the background uptake, specific ¹²⁵I-T₁AM uptake increased with increasing pH (Fig. 1E), suggesting that T₁AM uptake is driven by an outwardly directed proton gradient (or inwardly directed hydroxide gradient). The observed increase in total and background T₁AM uptake at higher pH probably reflects enhanced passive diffusion because there would be an increased fraction of the deprotonated form of the monoamine, which is positively charged at physiological pH of 7.4.

Specificity of T₁AM uptake

As an initial attempt at identifying membrane transporters that are involved in T₁AM uptake, we performed competition experiments using prototypical substrates of several major classes of transporters. These unlabeled compounds were incubated with ¹²⁵I-T₁AM to examine the specificity of the T₁AM uptake mechanism. The monoamine neurotransmitters dopamine, serotonin, and norepinephrine are transported by both high-affinity transporters of the SLC6 family (9) as well as low-affinity, high-capacity transporters such as PMAT (15) and transporters of the SLC22 family (17). Thyroid hormone is also transported by a variety of transporters, including organic anion transporters, amino acid transporters, and the more thyroid hormone-specific MCT8 (13). The biogenic amines dopamine, serotonin, and norepinephrine, as well as the thyroid hormone T₃, were added during ¹²⁵I-T₁AM incubation, and no competition for uptake was observed (Fig. 2A). These observations suggest that T₁AM is not taken up into the cell via the same mechanism as these other compounds.

Figure 2.

Specificity of T₁AM transport. A, Effect of other biogenic amines and thyroid hormone on T₁AM uptake. T₁AM uptake is not inhibited by the presence of excess unlabeled dopamine (DA), serotonin (5-HT), norepinephrine (NE), or thyroid hormone (T₃), showing that uptake is specific to T₁AM. B, Effect of other compounds on T₁AM uptake. T₁AM uptake is not significantly or dose-dependently inhibited by other substrates and inhibitors of the OCT/OAT/ENT transporter families, suggesting that T₁AM uptake does not occur via these transporters. 1-Methyl-4-phenylpyridinium (MPP⁺) is a prototypical organic cation and substrate of the OCT family of transporters as well as PMAT, and paraaminohippurate (PAH) is a prototypical organic anion and substrate of the OAT family of transporters. The nucleoside adenosine is a substrate of the ENT family of transporters. Corticosterone is an OCT inhibitor and decynium is an OCT and PMAT inhibitor. In both graphs, relative ¹²⁵I-T₁AM uptake values are normalized to the maximum uptake signal (disintegrations per minute radioactivity counts) obtained with ¹²⁵I-T₁AM incubation alone.

Moreover, several known substrates and inhibitors of the class of organic cation transporters (OCTs), organic anion transporters (OATs), and the equilibrative nucleoside transporters (ENTs), which include PMAT (ENT4), were also added at concentrations significantly greater than their observed Michaelis constant or inhibition constant values (15,17) and did not inhibit the uptake of T₁AM in a dose-dependent manner (Fig. 2B), suggesting that T₁AM uptake does not occur through these transporters.

Testing of potential candidate transporters for uptake of T₁AM

Our next step in identifying T₁AM transporters was a rational candidate-based approach. Despite the observed lack of competition of T₁AM uptake by prototypical substrates of certain transporter families, it is still conceivable that T₁AM may be a substrate of one of these transporters but that the existence of multiple T₁AM transporters could mask the effects of these substrates. Our pharmacological characterization of T₁AM uptake revealed sodium and chloride independence, which is a property shared by the SLC22 family of transporters (17) as well as by PMAT (SLC29A4) (15) and the thyroid hormone transporter MCT8 (SLC16A2) (18). Additionally, as an organic cation, T₁AM is a logical candidate substrate of the polyspecific organic ion transporters of the SLC22 family. Being a monoamine, T₁AM could likewise be transported by PMAT, and because of its close structural similarity to thyroid hormone, might be a substrate of MCT8. Thus, we directly tested several of these transporters for uptake of T₁AM using stable cell lines overexpressing the transporters. Whereas the stable cell lines exhibited increased uptake of their respective control substrates, none of these tested candidate transporters displayed increased uptake of T₁AM relative to empty vector stably transfected cells (Fig. 3). Together with the competition experiments performed, these direct transport studies strongly suggest that some of the most likely organic ion and monoamine transporters are not responsible for T₁AM uptake and instead a unique mechanism is involved. Identification of T₁AM transporters thus requires a more general approach, as the transporters may be previously characterized transporters of unrelated compounds or orphan transporters.

Figure 3.

Uptake of control substrates and T₁AM for several transporters. A selection of logical candidate transporters were tested for uptake of T₁AM in FlpIn HEK 293 cells (or MDCK for PMAT) stably expressing the transporter. The control substrates tested were 1-methyl-4-phenylpyridinium (OCT1, OCT2, OCT3, PMAT), tetraethylammonium (OCTN1), carnitine (OCTN2), paraaminohippurate (OAT1), acyclovir (OAT2), estrone sulfate (OAT3, OAT4), and T₃ (MCT8). Relative to empty vector-transfected cells, an increased uptake of at least 4-fold was observed for the control substrates, but no significant increase in uptake was found for T₁AM. For each substrate, uptake values are normalized to the average uptake signal measured for that particular substrate in the empty vector-transfected cells.

Development of an RNA interference (RNAi) screening method to identify T₁AM transporters

After a rational candidate-based approach did not identify a T₁AM transporter, we adopted a more unbiased, large-scale approach, using a library of siRNAs against 403 membrane transporters consisting of all of the SLC series of transporters characterized at this time, including pseudogenes and orphan transporters associated with this superfamily. The goal was to identify transporters that, when knocked down, resulted in decreased uptake of T₁AM. Table 1 lists the 46 families of transporters targeted by the siRNA library. The siRNAs were designed and constructed by Ambion and received as a custom transporter siRNA library. Three different siRNAs per target, for a total of 1209 unique siRNAs, were individually transfected into HeLa cells and T₁AM uptake was measured 48 h after transfection under the standard uptake conditions used previously. Figure 4A shows the experimental workflow of the RNAi screen. Transporters identified as positive hits were those for which at least two of the three siRNA probes against that particular transporter target resulted in a 30% or greater reduction in T₁AM uptake when compared with cells transfected with a negative control siRNA targeting no specific part of the genome. A 30% reduction in T₁AM uptake corresponds to a reduction level that is 1.5 sd greater than the mean reduction in uptake observed for all 1209 siRNAs. A positive control of siRNA against GAPDH was used to confirm successful transfection and gene knockdown. For each set of transfection experiments, the positive GAPDH siRNA control transfection resulted in at least 70% knockdown of GAPDH mRNA as determined by qRT-PCR.

Table 1.

The 46 families of 403 transporters in the HUGO solute carrier series

Transporter families
SLC1: The high-affinity glutamate and neutral amino acid transporters	SLC24: The Na+/(Ca2+-K+) exchangers
SLC2: The facilitative GLUT transporters	SLC25: The mitochondrial carriers
SLC3: The heavy subunits of the heteromeric amino acid transporters	SLC26: The multifunctional anion exchangers
SLC4: The bicarbonate transporters	SLC27: The fatty acid transport proteins
SLC5: The sodium glucose cotransporters	SLC28: The Na+-coupled nucleoside transporters
SLC6: The Na+- and Cl−-dependent neurotransmitter transporters	SLC29: The facilitative nucleoside transporters
SLC7: The cationic amino acid transporter/glycoprotein-associated	SLC30: The zinc efflux proteins
SLC8: The Na+/Ca2+ exchangers	SLC31: The copper transporters
SLC9: The Na+/H+ exchangers	SLC32: The vesicular inhibitory amino acid transporter
SLC10: The sodium bile salt cotransporters	SLC33: The acetyl-CoA transporter
SLC11: The proton-coupled metal ion transporters	SLC34: The type II Na+-phosphate cotransporters
SLC12: The electroneutral cation-Cl− cotransporters	SLC35: The nucleoside-sugar transporters
SLC13: The human Na+-sulfate/carboxylate cotransporters	SLC36: The proton-coupled amino acid transporters
SLC14: The urea transporters	SLC37: The sugar-phosphate/phosphate exchangers
SLC15: The proton oligopeptide cotransporters	SLC38: The system A & N, Na+-coupled neutral amino acid transporters
SLC16: The monocarboxylate transporters	SLC39: The metal ion transporters
SLC17: The vesicular glutamate transporters	SLC40: The basolateral iron transporter
SLC18: The vesicular amine transporters	SLC41: The MgtE-like magnesium transporters
SLC19: The folate/thiamine transporters	SLC42: The Rh ammonium transporters
SLC20: The type III Na+-phosphate cotransporters	SLC43: The Na+-independent, system L-like amino acid transporters
SLC21/SLCO: The organic anion transportins	SLC44: The choline-like transporters
SLC22: The organic cation/anion/zwitterion transporters	SLC45: The putative sugar transporters
SLC23: The Na+-dependent ascorbic acid transporters	SLC46: The heme transporters

Preliminary transporter candidates resulting from siRNA screening
SLC6A18	Neurotransmitter transporter	SLCO6A1	Organic anion transporter
SLC7A1	Cationic amino acid transporter	SLC26A11	Anion exchanger
SLC7A2	Cationic amino acid transporter	SLC27A1	Fatty acid transporter
SLC7A14	Cationic amino acid transporter	SLC28A3	Concentrative Na+-nucleoside transporter
SLC9A4	Sodium/hydrogen exchanger	SLC29A2	Equilibrative nucleoside transporter
SLC9A5	Sodium/hydrogen exchanger	SLC30A8	Zinc transporter
SLC9A6	Sodium/hydrogen exchanger	SLC30A10	Zinc transporter
SLC9A7	Sodium/hydrogen exchanger	SLC31A1	Copper transporter
SLC9A8	Sodium/hydrogen exchanger	SLC35C2	Ovarian cancer overexpressed 1
SLC9A9	Sodium/hydrogen exchanger	SLC35D2	UDP-N-acetylglucosamine transporter
SLC16A7	Monocarboxylate transporter	SLC37A1	Glycerol-3-phosphate transporter
SLC17A5	Anion/sugar transporter	SLC42A2	Rhesus blood group, B glycoprotein
SLCO1A2	Organic anion transporter	SLC42A3	Rhesus blood group, C glycoprotein
SLCO3A1	Organic anion transporter	SLC43A3	System L-like amino acid transporter
SLCO4A1	Organic anion transporter	SLC45A2	Putative sugar transporter
SLCO4C1	Organic anion transporter	SLC45A4	Putative sugar transporter
SLCO5A1	Organic anion transporter	SLC46A1	Heme transporter

Preliminary candidates consisted of 34 transporters of the 403 included in the siRNA library, giving an 8% hit rate. Only 12 of these transporters (SLC7A1, SLC16A7, SLC17A5, SLCO3A1, SLCO4A1, SLC29A2, SLC31A1, SLC43A3, SLC9A6, SLC9A9, SLC35C2, and SLC35D2) are expressed in HeLa cells, and four of these (SLC9A6, SLC9A9, SLC35C2, and SLC35D2) are expressed only intracellularly rather than at the plasma membrane. The final remaining eight transporter candidates are shown in bold. 

Figure 4.

RNAi screen design and resulting candidates. A, Experimental scheme for high-throughput screening of the transporter siRNA library to identify transporters involved in uptake of T₁AM. The siRNA library used for the screen consisted of 403 total transporter targets, including all SLC series transporters, with three distinct siRNAs per target. A total of 1209 unique siRNAs were individually transfected into HeLa cells using NeoFX transfection agent, and the transfected cells were assayed for uptake of T₁AM 48 h after transfection. A positive hit was defined as a target for which at least two of the three siRNAs produced a 30% or greater reduction in T₁AM uptake, when compared with cells transfected with negative control siRNA; these hits comprised the preliminary transporter candidates. After analysis of endogenous gene expression levels and cellular localization (plasma membrane vs. vesicular/mitochondrial membrane), the transporter siRNAs were retested and the candidate transporters were reduced to a subset of eight transporters. Future studies are required to validate the transporter candidates through an approach such as overexpression in cell lines and direct measurement of T₁AM uptake. B, Average levels of T₁AM uptake in HeLa cells transfected with siRNAs targeting the final eight transporter candidates identified in the screen, expressed relative to uptake in cells transfected with negative control (Neg.) siRNA. Each value represents the mean of triplicate determinations with variations of 1–6%.

Using these initial criteria, 34 transporters were identified (Table 1, bottom). To eliminate potential false-positive hits, gene expression levels of the transporters in HeLa cells were determined experimentally by RT-PCR (supplemental Fig. 1, published as supplemental data on The Endocrine Society's Journals Online web site at http://endo.endojournals.org) and verified using previously published microarray data from The Genomics Institute of the Novartis Foundation (19); those transporters not expressed in HeLa cells were discarded. Of the 12 transporters remaining, only eight are expressed at the plasma membrane as opposed to intracellular vesicles, based on Entrez Gene, a National Center for Biotechnology Information database (Bethesda, MD) of gene-specific and general protein information (20). The final list of transporters both expressed in HeLa cells and localized to the plasma membrane are highlighted in bold in Table 1 and included several organic ion transporters, a nucleoside transporter, an amino acid transporter, a monocarboxylate transporter, a copper transporter, and an orphan transporter belonging to the SLC43 family. The average levels of T₁AM uptake resulting from transfection with the siRNAs against these transporters is displayed in Fig. 4B. These eight transporters were retested at least three times, and the data shown depict average values from a representative experiment with each condition performed in triplicate. In addition, we directly measured levels of gene knockdown of these eight transporters, rather than relying on only a GAPDH siRNA-positive transfection control. Knockdown efficiency of these transporters as determined by qRT-PCR varied among the different transporters but was typically at least 50–60%, rather than the 70% or greater knockdown observed for GAPDH (Fig. 5), which is not unexpected given the extent of revalidation of the commercially available GAPDH siRNA, as opposed to the previously unvalidated efficacy of the transporter siRNAs.

Figure 5.

Expression of GAPDH and membrane transporters after siRNA transfection. The mRNA levels of GAPDH after positive control GAPDH siRNA transfection were determined by qRT-PCR and used to confirm successful transfection and gene knockdown for each set of transfections performed during transporter siRNA library screening. GAPDH expression was normalized to PGK1, and for each transfection experiment, at least 70% knockdown of GAPDH mRNA was observed. After obtaining the final eight transporter candidates, qRT-PCR was used to determine levels of gene knockdown after siRNA transfection for each of these specific transporters and was typically at least 50–60%, although knockdown efficiency varied among the different transporters. Transporter expression levels were normalized to GAPDH in cells transfected only with transporter siRNA, and the levels obtained after transfection with each of the three siRNAs targeting the particular transporter were averaged. All expression levels are shown relative to the expression of the corresponding target gene in cells transfected with negative control (Neg.) siRNA and were calculated using the comparative Ct method.

To examine the effect of overexpressing the transporters identified in the RNAi screen, we cloned each of the transporters and transfected them into FlpIn HEK cells. One transporter, SLC29A2, is unlikely to be a T₁AM transporter because the uptake of the control substrate, inosine, was enhanced, whereas the uptake of T₁AM was not. However, we were unable to use overexpression to confirm or refute the results of the RNAi screen for the remaining seven transporters. Five of the seven transporters (SLC7A1, SLC16A7, SLC31A1, and the OATPs SLCO3A1 and SLCO4A1) are abundantly expressed in multiple cell lines including HEK cells (19); therefore, their activities are difficult to measure over the background, and we did not observe increased uptake of various prototypical substrates or of T₁AM. In an attempt to find a lower background system, MDCK and CHO stable cell lines were constructed or obtained for these transporters, but they also did not exhibit a reproducible increase in uptake of T₁AM or any control substrate. Additionally, HEK 293T and COS-1 cells were transiently transfected with the transporter constructs in an attempt to obtain higher, although temporary, expression levels and a resulting increase in function, but these likewise displayed no significant enhancement of substrate uptake. SLC43A3 is an orphan transporter of the sodium-independent, System L-like amino acid transporters, and interestingly is highly expressed in the thyroid, but the SLC43A3 HEK FlpIn stable cell line failed to show increased uptake of any compound tested, including T₁AM, T₃, T₄, tyrosine, and phenylalanine. An obvious challenge in studying an orphan transporter like SLC43A3 is the lack of a known substrate with which to test functionality of the stable cell line. We constructed a SLC43A3-GFP stable cell line in parallel and, whereas we did observe clear plasma membrane localization of the transporter (data not shown), suggesting functional expression, we did not identify any substrate of this transporter.

Discussion

T₁AM is a recently discovered endogenous metabolite of thyroid hormone with dramatic physiological actions when administered in vivo. In the current study, specific cellular uptake of T₁AM was observed in a variety of cultured cell lines, suggesting a ubiquitous transport mechanism consistent with the widespread tissue accumulation of T₁AM and its wide range of actions, including hypothermia, bradycardia, hyperglycemia, and general behavioral inactivity. Although multiple transporters throughout the body likely contribute to intracellular accumulation of T₁AM, the uptake mechanism is relatively specific. Because thyronamines are the only molecules found to compete with T₁AM for uptake, it appears that the cellular uptake mechanism of T₁AM is specific for certain thyronamines and is distinct from that of the classical monoamine neurotransmitters, thyroid hormone, and other organic ions. Nearly all of the other thyronamines inhibited uptake of T₁AM but varied somewhat in potency. The specific iodination states of the thyronamines, therefore, are likely to be important to some degree for uptake. Interestingly, the only thyronamine not found to compete with T₁AM for uptake, T₄AM, was also the only other member of the thyronamine compounds that did not display inhibitory activity against the vesicular monoamine transporter VMAT2 (7).

In an attempt to identify plasma membrane transporters responsible for the uptake of T₁AM, we developed a high-throughput RNAi screening method in which a library of siRNAs targeting all of the solute carrier series of membrane transporters was transfected into HeLa cells and the siRNAs producing the greatest degree of reduction of T₁AM uptake were identified. The transporters targeted by these siRNAs are likely to be involved in T₁AM uptake into cells. A total of 34 of 403 transporters were initially identified as facilitating T₁AM uptake in HeLa cells. The 34 included several heavy metal transporters and various inorganic and organic ion transporters. As would have been expected, none of the likely candidate transporters previously tested and ruled out as T₁AM transporters displayed reduced T₁AM uptake after siRNA transfection. After examining endogenous expression levels in HeLa cells and cellular localization of the 34 transporters, we obtained a list of eight transporters that were retested and consistently displayed decreased T₁AM uptake function when knocked down.

Direct testing of transporters identified by the RNAi screen, however, was inconclusive. Even though decreased T₁AM uptake was observed when several transporters were knocked down in HeLa cells, overexpression of these transporters did not show the expected increase in T₁AM uptake. One of the challenges posed by the particular transporter candidates resulting from the RNAi screen included high background expression of the transporter, making it difficult to detect an increase in substrate uptake over an already high level. On the other hand, the lack of a control substrate for the orphan transporter SLC43A3, which has relatively low expression in HEK cells, made it difficult to determine whether the stable cell line created was indeed functional or whether experiments were being performed under optimal uptake assay conditions particular to this transporter.

Moreover, some transporter candidates identified by the RNAi screen might in fact be the result of indirect effects. Of the 34 initial candidates, only eight transporters are expressed in HeLa cells and localized to the plasma membrane. The high rate of false positives is likely due to secondary effects, such as cellular toxicity or disruption of membrane integrity. In addition, knocking down a transporter may affect the cellular content of another substance that influences the activity of the true transporter directly responsible for ligand uptake. For instance, SLC16A7 is a lactate transporter, and although it cannot be ruled out as a T₁AM transporter (indeed, the thyroid hormone transporter MCT8 is an SLC16 family member), this transporter may affect the intracellular ion content and pH of cells and consequently alter T₁AM uptake.

Nevertheless, the RNAi screening method developed here for T₁AM is a broadly applicable approach to potentially identify all transporters involved in the uptake of any particular compound in a particular cell type or tissue. Unlike the use of expression cloning to identify a particular gene responsible for activity, such an RNAi screen is advantageous because it could in theory be used even in a system in which there is high background uptake of the compound of interest because it relies on the knockdown of the activity rather than the enhancement over background signal. This inhibitory screening method and the traditional technique of expression cloning can be mutually complementary approaches with their own benefits, depending on the circumstances. For instance, when there is a source of high activity compared with the expression system, a cDNA library is likely to provide an enriched pool of genes responsible for the activity, which is a major advantage in expression cloning. However, when all available sources exhibit similar levels of activity, it might be more difficult to construct a complete cDNA library representing each gene at a sufficiently high level for functional detection. In this case, a high-throughput siRNA library screen can be more technically feasible. First, rather than testing every gene of the genome, the set of genes examined can be specified to a certain subset of interest, in this case membrane transporters. In addition, the growing popularity of RNAi methods is resulting in an improvement in siRNA design algorithms used in the creation of commercially available library collections. Finally, large numbers of siRNAs can be screened relatively quickly with optimized transfection conditions, followed by functional assessment.

Many endogenous compounds and xenobiotics have multiple transporters responsible for their uptake into cells. For example, although thyroid hormones had been originally thought to enter target cells by passive diffusion, several transport mechanisms are now known to be responsible for their uptake. A broad range of transporter types mediate intracellular entry of thyroid hormones, including monocarboxylate transporters, amino acid transporters, and classic multispecific organic anion/cation transporters such as several OATP family members (21,22,23). The transport of thyroid hormones into their target tissues by saturable mechanisms is critical for proper physiological control of both their action and metabolism. Thus, to gain an understanding of the physiological function and regulation of T₁AM, it is necessary to study its transport mechanisms into cells. The discovery of a specific transport mechanism for T₁AM presented in this study provides important additional insight into the role of this relatively new class of signaling molecules, although further elucidation of the particular transporters involved is required.

In conclusion, we have demonstrated that there exists specific intracellular transport of T₁AM, an important endogenous metabolite of thyroid hormone with physiological actions opposite those of its precursor and with previously demonstrated functions as a neuromodulator. Identification of the particular transporters responsible for T₁AM uptake would provide additional insight into its mechanism of action and specific biological roles, but these transporters currently remain unknown. Using a novel RNAi screening method, we have identified eight transporters that when knocked down reproducibly result in reduced T₁AM transport in HeLa cells. It is possible that these transporters collectively participate in the regulation of intracellular levels of T₁AM. On the other hand, transport of T₁AM may also be mediated by a non-SLC transporter because novel transporter genes are continually being discovered. Alternatively, a distinct transport mechanism altogether may be involved. For example, megalin has been identified as an endocytic receptor for the cellular uptake of steroid hormones including vitamin D, androgens, and estrogens, although megalin can act as a receptor for a wide variety of ligands (24). Potential T₁AM transport mechanisms thus include specific membrane uptake transporters and receptor-mediated endocytosis. These mechanisms could serve in regulation of T₁AM action in several ways. The action of T₁AM at extracellular targets such as TAAR1 may be terminated by its uptake into the cell, similar to the reuptake mechanisms for the monoamine neurotransmitters. Passage of T₁AM into the cell may also serve to provide access to intracellular targets, such as the vesicular monoamine transporter VMAT2, or to perform intracellular functions currently unknown. An additional motivation for identifying transporters of T₁AM is the potential elucidation of certain endocrine or neurological pathologies. Just as thyroid hormone transporter dysfunction has clearly been linked to particular disorders, such as MCT8 mutations leading to X-linked psychomotor retardation, improper functioning of T₁AM transporters could also lead to disease syndromes. Further studies into the recently discovered transport mechanism of T₁AM described here for the first time would be invaluable in our understanding of not only T₁AM action but also its potential implications for thyroid hormone regulation and possible involvement in thyroid-related pathologies.