Possible Involvement of Organic Anion Transporting Polypeptide 1c1 in the Photoperiodic Response of Gonads in Birds
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《内分泌学杂志》
Division of Biomodeling, Graduate School of Bioagricultural Sciences (N.N., T.Ta., S.Y., S.E., T.Yo.), Institute for Advanced Research (T.Yo.), Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan
Department of Applied Biochemistry, Faculty of Agriculture (M.I., T.M., T.Ya.), Genomics Research Institute (T.Ts.), Utsunomiya University, Mine-machi, Utsunomiya, Tochigi 321-8505, Japan
Abstract
The photoperiodic response of the gonads requires T3, which is generated photoperiodically from T4 by type 2 iodothyronine deiodinase in the hypothalamus. Although thyroid hormones were long thought to traverse the plasma membrane by passive diffusion due to their lipophilic nature, it is now known that several organic anion transporting polypeptides (Oatp) transport thyroid hormones into target cells. In this study, we have used database searches to isolate DNA sequences encoding members of the chicken Oatp family and constructed a molecular phylogenetic tree. Comprehensive expression analyses using in situ hybridization revealed strong expression of cOatp1c1 and weak expression of cOatp1b1 in the ventro-lateral walls of basal tuberal hypothalamus, whereas expression of four genes (cOatp1a1, cOatp1b1, cOatp1c1, and cOatp3a2) was observed in the choroid plexus. Expression levels of all these genes in both regions were not different between short-day and long-day conditions. Functional expression of cOatp1c1 in Chinese hamster ovary cells revealed that cOatp1c1 is a highly specific transporter for T4 with an apparent Km of 6.8 nM and a Vmax of 1.50 pmol per milligram of protein per minute. These results suggest that cOatp1c1 could be involved in the thyroxine transport necessary for the avian photoperiodic response of the gonads.
Introduction
MOST BIRDS LIVING outside the tropics use changes in day length to time their breeding seasons. In a previous study, we showed that T3 generated photoperiodically by type 2 iodothyronine deiodinase (Dio2) in the hypothalamus is critical for the photoperiodic response of the gonads in Japanese quail (1). Although thyroid hormones were long thought to traverse the plasma membrane by passive diffusion due to their lipophilic nature, it is now believed that a membrane transport system for thyroid hormones exists. Organic anion transporting polypeptides (Oatp) belong to solute carrier family 21. They form the Oatp/Slco superfamily of sodium-independent transport systems that mediate transmembrane transport of a wide range of endogenous and exogenous, amphipathic organic compounds (2, 3). An increasing number of studies in mammals have shown that some of the members are involved in the thyroid hormone transport system. In contrast to mammals, no study of avian Oatps has been reported to date. The draft sequence of the chicken genome (4) has finally allowed the analysis of Oatp/Slco genes in birds. Therefore, in the present study, we first isolated DNA sequences of the chicken Oatp family using database searches and constructed a molecular phylogenetic tree. Although chicken shows a robust photoperiodic response (5), the Japanese quail is more convenient and is more frequently used than chicken for studying avian photoperiodism due to its small size. Because chicken (Gallus gallus) and quail (Coturnix japonica) belong to the same order galliformes, and nucleotide sequences between the two species are highly conserved; accordingly, chicken and quail probes are widely used for both species when using methods such as Northern hybridization, Southern hybridization, in situ hybridization, and chromosomal mapping using fluorescent in situ hybridization (6, 7, 8, 9, 10). Comprehensive expression analysis of the Oatp family in the Japanese quail hypothalamus was performed using in situ hybridization. Among the Oatp family, expression of four genes (cOatp1a1, cOatp1b1, cOatp1c1, and cOatp3a2) was observed in the choroid plexus. In the ventro-lateral walls of the basal tuberal hypothalamus (BTH), strong expression of cOatp1c1 and weak expression of cOatp1b1 were observed. In both regions, expression levels of all genes were not different between short-day (SD) and long-day (LD) conditions. Finally, studies with stably expressed functional proteins in Chinese hamster ovary (CHO) cells indicate that cOatp1c1 is a high-affinity thyroid hormone transporter that could be involved in the photoperiodic response of the gonads.
Materials and Methods
Homology analysis
A database search was performed to obtain chicken Oapt genes using the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) and the Ensembl (http://www.ensembl.org/) databases. Multiple sequence alignments of amino acid sequences and phylogenetic tree construction were carried out using the ClustalW program (http://www.ebi.ac.uk/clustalw/) (11). The phylogenetic tree was visualized using TreeView (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html) (12).
Animals
Male, 4-wk-old Japanese quail were obtained from a local dealer and kept under SD conditions (8L:16D) at 24 ± 1 C in light-tight boxes (55 x 210 x 62 cm). Light was supplied by fluorescent lamps with a light intensity of 200 lux measured at the level of the bird’s head. SD birds were kept under SD conditions throughout the experiment and LD birds were transferred from SD to LD (16L:8D) at 7 wk old for 2 wk. Testicular weight of LD birds (968 ± 164 mg) and SD birds (287 ± 14.8 mg) was significantly different (Mann Whitney U test, P < 0.05). Food and water were provided ad libitum. Birds were treated in accordance with the guidelines of Nagoya University.
In situ hybridization
At the age of 9 wk, quail were killed by decapitation, and the brains were removed immediately to avoid acute changes in gene expression. In situ hybridization was carried out as previously described (10). Antisense and sense 45-mer oligonucleotide probes were labeled with [-33P]dATP (NEN Life Science Products, Boston, MA) using-terminal deoxyribonucleotidyl transferase (Invitrogen Life Technologies, Inc., Gaithersburg, MD).
cOatp1a1 (GenBank accession no.: XM_416421): 5'-ccggttcctgttgaggccttacacccagccagacacgatgacaca-3'; cOatp1b1 (XM_416418): 5'-accaagccaccaggcacccacccagcgagagtcctgtggagtgat-3'; cOatp1c1 (AB206652): 5'-gacacctactgacaaagtgtatgaagtgataaccgatattaccag-3'; cOatp2a1 (XM_422673): 5'-ccaatgtccacaaagagccgcagcaccacagagcccagcaggtac-3'; cOatp2b1 (XM_417242): 5'-gtgttccccacctcgttgaaggacgcgagcagccccgacgtcttg-3'; cOatp3a1 (XM_413876): 5'-gtcccacagctctctgccacccctgctaatctctccagttcccac-3'; cOatp3a2 (XM_416419): 5'-gtacaagacacagcaccctgcccgtagtcccgcaatgaggccaag-3'; cOatp4a1 (XM_417407): 5'-agaggcaggccgcgatgtcgtaggagctggcgatcagcccgctct-3'; cOatp5a1 (XM_418287): 5'-tggcccactgtcggtggggtgataacctggcggctttggacacag-3'; cOatp5a2 (XM_418288): 5'-tcgaagcagctgaccagcagccccgactccgagctcttcaggctg-3'.
Hybridization was carried out overnight at 42 C. After the glass slides were washed, they were air dried and exposed to Biomax-MR film (Eastman Kodak Co., New York, NY) for 2 wk with 14C standards (American Radiolabeled Chemicals, St. Louis, MO). Relative optical densities were measured by using a computed image-analyzing system (MCID Imaging Research, St. Catharines, Ontario, Canada), and were converted into a relative radioactive value (nanocuries) using 14C standards. Specific hybridization signals were obtained by subtracting background values obtained from adjacent brain areas that did not exhibit a hybridization signal.
Cloning of cOatp1c1 cDNA
Total RNA was extracted from the mediobasal hypothalamus (MBH) of a 6-month-old egg-laying hen by using TRIzol Reagent (Invitrogen). cDNA synthesis was performed using total RNA primed with oligo(dT) using SuperScript III (Invitrogen) at 50 C for 60 min. cOatp1c1 cDNA was amplified by PCR using TaKaRa Ex Taq (Takara, Otsu, Japan) with sense 5'-CCCAAGCTTACCATGGAGATTTCTTCAAAA-3' and antisense 5'- GGGTTTAAACCTAAAGCCTAGTCTCCTTTT-3' primers derived from the predicted cOatp1c1 sequence (HindIII and PmeI site sequences are underlined and the Kozak sequence (13) is in bold) under the following cycle conditions: 30 cycles of 95 C (20 sec), 60 C (30 sec), 72 C (3 min) for amplification, followed by 72 C (10 min) for the final extension. The amplified cDNA fragment was digested with HindIII-PmeI and this fragment was subcloned into the pcDNA3.1 expression vector (Invitrogen), which had been digested with HindIII-PmeI. To eliminate PCR errors, we sequenced 20 cDNA clones.
Stable expression of cOapt1c1 in CHO cells
CHO cells were cultured in Ham’s F12 (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen). A total of 1 x 105 cells were grown in a 6-cm dish and transfected with 7 μg cOatp1c1 expression vector using a modified calcium phosphate coprecipitation method (14). Sixteen hours after transfection, the medium was changed twice within a 1-h interval. Cells were further cultured for 1 d and replated into a 10-cm dish. Stable transformants were selected with 600 mg/ml G418 (Sigma, St. Louis, MO) for 2 d and then with 400 mg/ml G418 for 8 d. Surviving cells were pooled, and eighteen clones were isolated by the limiting dilution method. Two of the eighteen clones showed significant incorporation of [125I]T4 (NEN Life Science Products), and then one clone named 2d was used for further study. Similarly, three stable clones of cells transfected with pcDNA3.1 empty vector were isolated, and clone 1a was used as a negative control.
Transport study
After cells (1 x 105 cells per well) were washed three times and preincubated with Krebs-Henseleit buffer at 37 C for 15 min, uptake of [125I]T4 (NEN Life Science Products), [125I]T3 (NEN Life Science Products), or [125I]rT3 (Amersham Biosciences, Tokyo, Japan) was initiated by adding radiolabeled ligands in the buffer in the presence and absence of competitors (see Results; all from Sigma). The Krebs-Henseleit buffer consisted of 142 mM NaCl, 23.8 mM NaHCO3, 4.83 mM KCl, 0.96 mM KH2PO4, 1.20 mM MgSO4, 12.5 mM HEPES, 5 mM glucose, and 1.53 mM CaCl2 adjusted to pH 7.4. The uptake was terminated at designated times by adding ice-cold Krebs-Henseleit buffer. Cells were then washed twice with 1 ml of ice-cold Krebs-Henseleit buffer, resuspended in 400 μl of 0.2 N NaOH, and transferred to test tubes. The radioactivity associated with the cells was determined with a -counter (Aloka, Tokyo, Japan). The protein content of the cell lysates was determined by the method of Bradford (15) with bovine -globulin as the standard.
Results
Phylogenetic analysis of chicken Oatps
The result of the phylogenetic analysis is shown in Fig. 1. Ten amino acid sequences were classified into the Oatp superfamily (40% amino acid sequence identity). Accession numbers for each protein sequence and proposed protein names are indicated (see Discussion).
Expression of Oatps in the brain
Among 10 Oatps, expression of cOatp1a1, cOatp1b1, cOatp1c1, and cOatp3a2 was observed in the quail choroid plexus (Fig. 2). No signal was detected in the other six genes (data not shown). No hybridization signal was observed in the sense controls (only a representative picture for cOatp1c1 is shown). When we focused on the MBH, which is believed to be the center that controls photoperiodism, strong expression of cOatp1c1 and weak expression of cOatp1b1 were observed in the quail BTH (Fig. 3). No signal was detected in other eight genes (data not shown). The intensity of the cOatp1c1 signal was approximately 100-fold higher than that of cOatp1b1. Expression of cOatp1c1 was especially strong in the ependymal cell layer lining the ventro-lateral walls of the third ventricle (Fig. 4). However, no expression was observed in the floor of the third ventricle. Relatively weak expression was also observed around the ependymal cell layer such as in the infundibular nucleus. Expression of cOatp1b1 was also observed in similar regions (Fig. 3). We have also confirmed expression of these genes in the chicken choroid plexus and the BTH (published as supplemental Fig. 1 on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). In both regions (BTH and choroid plexus), the expression level was not different in both SD and LD conditions (Fig. 5; only the results for the BTH are shown).
Functional analysis of cOatp1c1
Because cOatp1c1 was predominantly expressed in the BTH and the substrates for mammalian Oatp1b1 are reported to be bile salt and organic anions, we have focused on cOatp1c1 for further functional analyses.
Figure 6 shows the time course of uptake of [125I]T4 (A; 2.5 nM), [125I]T3 (B; 2.5 nM), and [125I]rT3 (C; 2.5 nM) by cOatp1c1-expressing CHO cells and control (pcDNA3.1 empty vector) CHO cells. The uptake of [125I]T4 by cOatp1c1-expressing cells was markedly greater than that of the control cells. However, there were no significant differences in the uptake of [125I]T3 and [125I]rT3 between the two cell types. Thus, T4 is the preferred substrate for cOatp1c1.
Next, we determined the kinetics of [125I]T4 transport in the above cells. As shown in Fig. 7, initial uptake rates (1 min) exhibited clear saturability with increasing substrate concentrations. The calculated apparent Km value was 6.8 nM and the Vmax was 1.50 pmol per milligrams of protein per minute. These results further substantiate the conclusion that cOatp1c1 represents a high affinity transporter for T4.
Finally, we examined the cis-inhibitory effect of thyroid hormones, their related compounds, and organic anions on the cOatp1c1-mediated uptake of [125I]T4 (Fig. 8). The compounds tested were premixed in the buffer containing [125I]T4 (2.5 nM), and the uptake of [125I]T4 into the cells was determined as mentioned above. rT3 (Ki = 0.182 μM), 3,3',5-triiodothyroacetic acid (Ki = 2.31 μM), T4 (Ki = 3.55 μM), and T3 (Ki = 4.27 μM) were potent inhibitors of cOatp1c1. Probenecid (Ki = 14.6 μM), estradiol-17-(-D-glucuronide) (Ki = 19.1 μM), 3,5-diiodo-L-thyronine (Ki = 96.4 μM), and digoxin (Ki = 140 μM) were moderate inhibitors. Estrone-3-sulfate and taurocholate exhibited less than 50% inhibition even at 10–4 M (Ki > 731 μM; data not shown). p-Aminohippuric acid (10–4 M), cimetidine (10–4 M), ouabain (10–4 M), tetraethylammonium (10–4 M), leucine (10–3 M), phenylalanine (10–3 M), tryptophan (10–3 M), and tyrosine (10–3 M) had no inhibitory effects on cOatp1c1-mediated transport.
Discussion
Microarray analysis of the Siberian hamster hypothalamus revealed that expression levels of thyroid hormone transporters such as transthyretin, albumin, and thyroxine binding globulin are different between SD and LD conditions (16). Therefore, we first focused on the expression of Transthyretin and Albumin genes in the present study. However, we could not detect expression of these genes in the hypothalamus of Japanese quail (data not shown) (the avian homolog of thyroxine binding globulin has not yet been identified). Because the expression of these three genes was only examined by microarray analysis and RT-PCR in the Siberian hamster (16), it may be difficult to compare the different results. However, it is possible that this discrepancy may be due to the differences in the avian and mammalian systems.
Although thyroid hormones were thought to traverse the plasma membrane by passive diffusion due to their hydrophobic nature, it is now known that in mammalian species some organic anion transporters transport thyroid hormones into target cells. However, there have been no reports of avian Oatps to date. The chicken genome project has finally made possible the analysis of avian Oatps, and the present study is the first comprehensive analysis of the avian Oatp/Slco superfamily. Using a database search, we have isolated 10 chicken Oatp genes. Hagenbuch and Meier classified Oatps according to their amino acid sequence identities (3). Oatps within the same family and subfamily share greater than or equal to 40% and greater than or equal to 60% amino acid sequence identities, respectively. Individual families are designated by numerals (e.g. Oatp1, Oatp2, etc.), and individual subfamilies are designated by letters (e.g. Oatp1a, Oatp1b, etc.). In some subfamilies, there are several individual gene products, and in such cases, additional numbering based on the chronology of identification is used (e.g. Oatp1a1, Oatp1a2, etc.). Although these criteria are clear and useful among mammalian species, it is difficult to apply the same criteria to nonmammalian species. Because complete genome information for chicken and other nonmammalian vertebrate species is not yet available, it is currently difficult to assign protein names and gene symbols. However, for convenience, we propose to use temporal nomenclature for avian homologs of the Oatp superfamily as shown in Fig. 1.
Among the 10 Oatps, expression of cOatp1a1, cOatp1b1, cOatp1c1, and cOatp3a2 were observed in the choroid plexus. These Oatps appear to mediate the transmembrane transport of a wide range of endogenous and exogenous amphipathic, organic compounds from the circulating blood to the brain. In the BTH, strong expression of cOatp1c1 and weak expression of cOatp1b1 were observed. Expression of these genes was especially strong in the ependymal cells lining the ventro-lateral walls of the third ventricle. However, no expression was observed in the floor of the third ventricle. Relatively weak expression was also observed around the infundibular nucleus. These regions correlate well with the expression sites of the gene encoding the thyroid hormone inactivating enzyme, type 3 iodothyronine deiodinase (Dio3), and partly correlated with the expression sites of the gene encoding the thyroid hormone activating enzyme, Dio2 (Fig. 4). In quail, the ependymal cells bordering the third ventricle in the BTH are divided into the dorsal, ventro-lateral, and ventral regions; the four types of tanycytes are classified using morphological and metabolic characteristics (17). Type 1 and 4 tanycytes are found in the dorsal ependymal cells and the ventral ependymal cells, respectively. Type 2 and 3 tanycytes are associated with the ventro-lateral ependymal cells. According to the detailed analysis by Sharp (17), tanycytes type 2 and 3 occur in the latero-ventral walls of the BTH dorsal to the posterior median eminence, whereas only type 2 occurs in this region above anterior median eminence. Although cOatp1c1, cOatp1b1, and Dio3 appear to be expressed in the tanycytes in the ventro-lateral region and Dio2 appears to be expressed in the tanycytes in the ventro-lateral and ventral regions, the exact cell that expresses these genes remains unresolved.
Because the signal intensity of cOatp1c1 was approximately 100-fold higher than that of cOatp1b1, and the predominant substrates for mammalian Oatp1c1 and Oatp1b1 are reported to be thyroid hormones and bile salts, respectively, we have focused on cOatp1c1 for further functional analyses. In vitro transport studies using cOatp1c1-expressing CHO cells confirmed that cOatp1c1 transported [125I]T4 with a Km of 6.8 nM and a Vmax of 1.50 pmol per milligram of protein per minute but not [125I]T3. These are consistent with the results obtained in human Oatp1c1 (also known as OATP-F) and rat and mouse Oatp1c1 (also known as Oatp14) (18, 19, 20). Contrary to the fact the rodent counterpart prefers [125I]rT3 as the substrate (18, 19), [125I]rT3 was not transported in avian system. Thus, 3,3',5,5'-iodination is important for efficient transport by cOatp1c1, whereas 3',5'-iodination is required for mammalian Oatp1c1 transport (18, 19). Competition of [125I]T4 uptake with thyroid hormone-related compounds and organic anions that are known to be transported by Oatps also demonstrated the specificity of cOatp1c1. The relative inhibitory activity demonstrated in the present study was consistent with that of mammals although the affinity to the compounds that were tested is higher than those observed in the case of their mammalian counterparts (18, 19, 20). Hence, cOatp1c1 is considered to be a highly specific transporter for T4. We concluded that cOatp1c1 is the avian ortholog of Oatp1c1 (OATP-F in humans or Oatp14 in rodents), and it plays an important role in the delivery of T4 for the generation of the bioactive metabolite T3. The reason for the difference in the substrate specificity between the chicken and mammalian Oatp1c1 remains unknown. Furthermore, although cold T3 significantly inhibited [125I]T4 uptake into the cells that expresses cOatp1c1 and the Ki value of T3 was found to be similar to that of T4, [125I]T3 itself is not a suitable substrate for cOatp1c1. Further studies are required to identify the structural basis for the difference in the substrate preference of Oatps in vertebrates.
The brain is separated from the bloodstream by the blood-brain barrier and the blood-cerebrospinal fluid (CSF) barrier, and the thyroid hormones in the circulating blood have to cross the barriers to exert their effects. cOatp1c1 and transthyretin expressed in the choroid plexus could transport thyroxine into the CSF from the bloodstream (1, 2, 18, 19), whereas cOatp1c1 that was expressed in the ventro-lateral walls of the BTH could play a role in the cellular uptake of thyroxine from the CSF, which is then converted intracellularly either into active T3 by Dio2 or inactive rT3 by Dio3.
In a previous study, we showed that in addition to T3, the level of T4 was also increased in the MBH under LD conditions. However, in the present study, expression levels of Oatps in both the choroid plexus and the BTH show no differences between LD and SD conditions. Although we cannot exclude completely the possibility that the protein level(s) of Oatp(s) or the expression level of an unidentified thyroid transporter may be different between LD conditions and SD conditions, our recent report seems provide an explanation for the increased T4 content under LD conditions (21). Within the BTH, high expression of Dio3 and low expression of Dio2 occurs under SD conditions, whereas under LD conditions, low expression of Dio3 and high expression of Dio2 occurs. Dio3 converts not only T3 but also T4 into inactive metabolites by inner-ring deiodination. Thus, high expression of Dio3 under SD conditions appears to actively eliminate T4 when it is not required (Fig. 9). This reciprocal expression of Dio2 and Dio3 appears to enable birds to fine-tune the thyroid hormone content of the MBH.
In conclusion, we have performed a comprehensive analysis of chicken Oatps. Expression of cOatp1c1 colocalized with Dio2 and Dio3, and cOatp1c1 was shown to transport T4. These results suggest that cOatp1c1 could be involved in the avian photoperiodic response of the gonads. Thyroid hormone plays a critical role in the development and plasticity of the central nervous system, and seasonal morphological changes in the neuro-glial interaction between the GnRH nerve terminals and glial endfeet in the median eminence of quail have been reported (22). To elucidate the molecular mechanism involved in these morphological changes, future studies on the identification of the cellular localization of thyroid hormone receptors and search for the target gene(s) of T3 action are required.
Acknowledgments
We thank Nagoya University Radioisotope Center for use of its facilities.
Footnotes
T.Yo. was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences and a Grant-in-Aid for Encouragement of Young Scientists from the Ministry of Education, Science, Sports and Culture.
First Published Online November 17, 2005
1 The first four authors contributed equally to this work.
Abbreviations: BTH, Basal tuberal hypothalamus; CHO, Chinese hamster ovary; CSF, cerebrospinal fluid; Dio2, type 2 iodothyronine deiodinase; Dio3, type 3 iodothyronine deiodinase; LD, long day; MBH, mediobasal hypothalamus; Oatp, organic anion transporting polypeptide; SD, short day.
Accepted for publication November 10, 2005.
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Department of Applied Biochemistry, Faculty of Agriculture (M.I., T.M., T.Ya.), Genomics Research Institute (T.Ts.), Utsunomiya University, Mine-machi, Utsunomiya, Tochigi 321-8505, Japan
Abstract
The photoperiodic response of the gonads requires T3, which is generated photoperiodically from T4 by type 2 iodothyronine deiodinase in the hypothalamus. Although thyroid hormones were long thought to traverse the plasma membrane by passive diffusion due to their lipophilic nature, it is now known that several organic anion transporting polypeptides (Oatp) transport thyroid hormones into target cells. In this study, we have used database searches to isolate DNA sequences encoding members of the chicken Oatp family and constructed a molecular phylogenetic tree. Comprehensive expression analyses using in situ hybridization revealed strong expression of cOatp1c1 and weak expression of cOatp1b1 in the ventro-lateral walls of basal tuberal hypothalamus, whereas expression of four genes (cOatp1a1, cOatp1b1, cOatp1c1, and cOatp3a2) was observed in the choroid plexus. Expression levels of all these genes in both regions were not different between short-day and long-day conditions. Functional expression of cOatp1c1 in Chinese hamster ovary cells revealed that cOatp1c1 is a highly specific transporter for T4 with an apparent Km of 6.8 nM and a Vmax of 1.50 pmol per milligram of protein per minute. These results suggest that cOatp1c1 could be involved in the thyroxine transport necessary for the avian photoperiodic response of the gonads.
Introduction
MOST BIRDS LIVING outside the tropics use changes in day length to time their breeding seasons. In a previous study, we showed that T3 generated photoperiodically by type 2 iodothyronine deiodinase (Dio2) in the hypothalamus is critical for the photoperiodic response of the gonads in Japanese quail (1). Although thyroid hormones were long thought to traverse the plasma membrane by passive diffusion due to their lipophilic nature, it is now believed that a membrane transport system for thyroid hormones exists. Organic anion transporting polypeptides (Oatp) belong to solute carrier family 21. They form the Oatp/Slco superfamily of sodium-independent transport systems that mediate transmembrane transport of a wide range of endogenous and exogenous, amphipathic organic compounds (2, 3). An increasing number of studies in mammals have shown that some of the members are involved in the thyroid hormone transport system. In contrast to mammals, no study of avian Oatps has been reported to date. The draft sequence of the chicken genome (4) has finally allowed the analysis of Oatp/Slco genes in birds. Therefore, in the present study, we first isolated DNA sequences of the chicken Oatp family using database searches and constructed a molecular phylogenetic tree. Although chicken shows a robust photoperiodic response (5), the Japanese quail is more convenient and is more frequently used than chicken for studying avian photoperiodism due to its small size. Because chicken (Gallus gallus) and quail (Coturnix japonica) belong to the same order galliformes, and nucleotide sequences between the two species are highly conserved; accordingly, chicken and quail probes are widely used for both species when using methods such as Northern hybridization, Southern hybridization, in situ hybridization, and chromosomal mapping using fluorescent in situ hybridization (6, 7, 8, 9, 10). Comprehensive expression analysis of the Oatp family in the Japanese quail hypothalamus was performed using in situ hybridization. Among the Oatp family, expression of four genes (cOatp1a1, cOatp1b1, cOatp1c1, and cOatp3a2) was observed in the choroid plexus. In the ventro-lateral walls of the basal tuberal hypothalamus (BTH), strong expression of cOatp1c1 and weak expression of cOatp1b1 were observed. In both regions, expression levels of all genes were not different between short-day (SD) and long-day (LD) conditions. Finally, studies with stably expressed functional proteins in Chinese hamster ovary (CHO) cells indicate that cOatp1c1 is a high-affinity thyroid hormone transporter that could be involved in the photoperiodic response of the gonads.
Materials and Methods
Homology analysis
A database search was performed to obtain chicken Oapt genes using the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) and the Ensembl (http://www.ensembl.org/) databases. Multiple sequence alignments of amino acid sequences and phylogenetic tree construction were carried out using the ClustalW program (http://www.ebi.ac.uk/clustalw/) (11). The phylogenetic tree was visualized using TreeView (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html) (12).
Animals
Male, 4-wk-old Japanese quail were obtained from a local dealer and kept under SD conditions (8L:16D) at 24 ± 1 C in light-tight boxes (55 x 210 x 62 cm). Light was supplied by fluorescent lamps with a light intensity of 200 lux measured at the level of the bird’s head. SD birds were kept under SD conditions throughout the experiment and LD birds were transferred from SD to LD (16L:8D) at 7 wk old for 2 wk. Testicular weight of LD birds (968 ± 164 mg) and SD birds (287 ± 14.8 mg) was significantly different (Mann Whitney U test, P < 0.05). Food and water were provided ad libitum. Birds were treated in accordance with the guidelines of Nagoya University.
In situ hybridization
At the age of 9 wk, quail were killed by decapitation, and the brains were removed immediately to avoid acute changes in gene expression. In situ hybridization was carried out as previously described (10). Antisense and sense 45-mer oligonucleotide probes were labeled with [-33P]dATP (NEN Life Science Products, Boston, MA) using-terminal deoxyribonucleotidyl transferase (Invitrogen Life Technologies, Inc., Gaithersburg, MD).
cOatp1a1 (GenBank accession no.: XM_416421): 5'-ccggttcctgttgaggccttacacccagccagacacgatgacaca-3'; cOatp1b1 (XM_416418): 5'-accaagccaccaggcacccacccagcgagagtcctgtggagtgat-3'; cOatp1c1 (AB206652): 5'-gacacctactgacaaagtgtatgaagtgataaccgatattaccag-3'; cOatp2a1 (XM_422673): 5'-ccaatgtccacaaagagccgcagcaccacagagcccagcaggtac-3'; cOatp2b1 (XM_417242): 5'-gtgttccccacctcgttgaaggacgcgagcagccccgacgtcttg-3'; cOatp3a1 (XM_413876): 5'-gtcccacagctctctgccacccctgctaatctctccagttcccac-3'; cOatp3a2 (XM_416419): 5'-gtacaagacacagcaccctgcccgtagtcccgcaatgaggccaag-3'; cOatp4a1 (XM_417407): 5'-agaggcaggccgcgatgtcgtaggagctggcgatcagcccgctct-3'; cOatp5a1 (XM_418287): 5'-tggcccactgtcggtggggtgataacctggcggctttggacacag-3'; cOatp5a2 (XM_418288): 5'-tcgaagcagctgaccagcagccccgactccgagctcttcaggctg-3'.
Hybridization was carried out overnight at 42 C. After the glass slides were washed, they were air dried and exposed to Biomax-MR film (Eastman Kodak Co., New York, NY) for 2 wk with 14C standards (American Radiolabeled Chemicals, St. Louis, MO). Relative optical densities were measured by using a computed image-analyzing system (MCID Imaging Research, St. Catharines, Ontario, Canada), and were converted into a relative radioactive value (nanocuries) using 14C standards. Specific hybridization signals were obtained by subtracting background values obtained from adjacent brain areas that did not exhibit a hybridization signal.
Cloning of cOatp1c1 cDNA
Total RNA was extracted from the mediobasal hypothalamus (MBH) of a 6-month-old egg-laying hen by using TRIzol Reagent (Invitrogen). cDNA synthesis was performed using total RNA primed with oligo(dT) using SuperScript III (Invitrogen) at 50 C for 60 min. cOatp1c1 cDNA was amplified by PCR using TaKaRa Ex Taq (Takara, Otsu, Japan) with sense 5'-CCCAAGCTTACCATGGAGATTTCTTCAAAA-3' and antisense 5'- GGGTTTAAACCTAAAGCCTAGTCTCCTTTT-3' primers derived from the predicted cOatp1c1 sequence (HindIII and PmeI site sequences are underlined and the Kozak sequence (13) is in bold) under the following cycle conditions: 30 cycles of 95 C (20 sec), 60 C (30 sec), 72 C (3 min) for amplification, followed by 72 C (10 min) for the final extension. The amplified cDNA fragment was digested with HindIII-PmeI and this fragment was subcloned into the pcDNA3.1 expression vector (Invitrogen), which had been digested with HindIII-PmeI. To eliminate PCR errors, we sequenced 20 cDNA clones.
Stable expression of cOapt1c1 in CHO cells
CHO cells were cultured in Ham’s F12 (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen). A total of 1 x 105 cells were grown in a 6-cm dish and transfected with 7 μg cOatp1c1 expression vector using a modified calcium phosphate coprecipitation method (14). Sixteen hours after transfection, the medium was changed twice within a 1-h interval. Cells were further cultured for 1 d and replated into a 10-cm dish. Stable transformants were selected with 600 mg/ml G418 (Sigma, St. Louis, MO) for 2 d and then with 400 mg/ml G418 for 8 d. Surviving cells were pooled, and eighteen clones were isolated by the limiting dilution method. Two of the eighteen clones showed significant incorporation of [125I]T4 (NEN Life Science Products), and then one clone named 2d was used for further study. Similarly, three stable clones of cells transfected with pcDNA3.1 empty vector were isolated, and clone 1a was used as a negative control.
Transport study
After cells (1 x 105 cells per well) were washed three times and preincubated with Krebs-Henseleit buffer at 37 C for 15 min, uptake of [125I]T4 (NEN Life Science Products), [125I]T3 (NEN Life Science Products), or [125I]rT3 (Amersham Biosciences, Tokyo, Japan) was initiated by adding radiolabeled ligands in the buffer in the presence and absence of competitors (see Results; all from Sigma). The Krebs-Henseleit buffer consisted of 142 mM NaCl, 23.8 mM NaHCO3, 4.83 mM KCl, 0.96 mM KH2PO4, 1.20 mM MgSO4, 12.5 mM HEPES, 5 mM glucose, and 1.53 mM CaCl2 adjusted to pH 7.4. The uptake was terminated at designated times by adding ice-cold Krebs-Henseleit buffer. Cells were then washed twice with 1 ml of ice-cold Krebs-Henseleit buffer, resuspended in 400 μl of 0.2 N NaOH, and transferred to test tubes. The radioactivity associated with the cells was determined with a -counter (Aloka, Tokyo, Japan). The protein content of the cell lysates was determined by the method of Bradford (15) with bovine -globulin as the standard.
Results
Phylogenetic analysis of chicken Oatps
The result of the phylogenetic analysis is shown in Fig. 1. Ten amino acid sequences were classified into the Oatp superfamily (40% amino acid sequence identity). Accession numbers for each protein sequence and proposed protein names are indicated (see Discussion).
Expression of Oatps in the brain
Among 10 Oatps, expression of cOatp1a1, cOatp1b1, cOatp1c1, and cOatp3a2 was observed in the quail choroid plexus (Fig. 2). No signal was detected in the other six genes (data not shown). No hybridization signal was observed in the sense controls (only a representative picture for cOatp1c1 is shown). When we focused on the MBH, which is believed to be the center that controls photoperiodism, strong expression of cOatp1c1 and weak expression of cOatp1b1 were observed in the quail BTH (Fig. 3). No signal was detected in other eight genes (data not shown). The intensity of the cOatp1c1 signal was approximately 100-fold higher than that of cOatp1b1. Expression of cOatp1c1 was especially strong in the ependymal cell layer lining the ventro-lateral walls of the third ventricle (Fig. 4). However, no expression was observed in the floor of the third ventricle. Relatively weak expression was also observed around the ependymal cell layer such as in the infundibular nucleus. Expression of cOatp1b1 was also observed in similar regions (Fig. 3). We have also confirmed expression of these genes in the chicken choroid plexus and the BTH (published as supplemental Fig. 1 on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). In both regions (BTH and choroid plexus), the expression level was not different in both SD and LD conditions (Fig. 5; only the results for the BTH are shown).
Functional analysis of cOatp1c1
Because cOatp1c1 was predominantly expressed in the BTH and the substrates for mammalian Oatp1b1 are reported to be bile salt and organic anions, we have focused on cOatp1c1 for further functional analyses.
Figure 6 shows the time course of uptake of [125I]T4 (A; 2.5 nM), [125I]T3 (B; 2.5 nM), and [125I]rT3 (C; 2.5 nM) by cOatp1c1-expressing CHO cells and control (pcDNA3.1 empty vector) CHO cells. The uptake of [125I]T4 by cOatp1c1-expressing cells was markedly greater than that of the control cells. However, there were no significant differences in the uptake of [125I]T3 and [125I]rT3 between the two cell types. Thus, T4 is the preferred substrate for cOatp1c1.
Next, we determined the kinetics of [125I]T4 transport in the above cells. As shown in Fig. 7, initial uptake rates (1 min) exhibited clear saturability with increasing substrate concentrations. The calculated apparent Km value was 6.8 nM and the Vmax was 1.50 pmol per milligrams of protein per minute. These results further substantiate the conclusion that cOatp1c1 represents a high affinity transporter for T4.
Finally, we examined the cis-inhibitory effect of thyroid hormones, their related compounds, and organic anions on the cOatp1c1-mediated uptake of [125I]T4 (Fig. 8). The compounds tested were premixed in the buffer containing [125I]T4 (2.5 nM), and the uptake of [125I]T4 into the cells was determined as mentioned above. rT3 (Ki = 0.182 μM), 3,3',5-triiodothyroacetic acid (Ki = 2.31 μM), T4 (Ki = 3.55 μM), and T3 (Ki = 4.27 μM) were potent inhibitors of cOatp1c1. Probenecid (Ki = 14.6 μM), estradiol-17-(-D-glucuronide) (Ki = 19.1 μM), 3,5-diiodo-L-thyronine (Ki = 96.4 μM), and digoxin (Ki = 140 μM) were moderate inhibitors. Estrone-3-sulfate and taurocholate exhibited less than 50% inhibition even at 10–4 M (Ki > 731 μM; data not shown). p-Aminohippuric acid (10–4 M), cimetidine (10–4 M), ouabain (10–4 M), tetraethylammonium (10–4 M), leucine (10–3 M), phenylalanine (10–3 M), tryptophan (10–3 M), and tyrosine (10–3 M) had no inhibitory effects on cOatp1c1-mediated transport.
Discussion
Microarray analysis of the Siberian hamster hypothalamus revealed that expression levels of thyroid hormone transporters such as transthyretin, albumin, and thyroxine binding globulin are different between SD and LD conditions (16). Therefore, we first focused on the expression of Transthyretin and Albumin genes in the present study. However, we could not detect expression of these genes in the hypothalamus of Japanese quail (data not shown) (the avian homolog of thyroxine binding globulin has not yet been identified). Because the expression of these three genes was only examined by microarray analysis and RT-PCR in the Siberian hamster (16), it may be difficult to compare the different results. However, it is possible that this discrepancy may be due to the differences in the avian and mammalian systems.
Although thyroid hormones were thought to traverse the plasma membrane by passive diffusion due to their hydrophobic nature, it is now known that in mammalian species some organic anion transporters transport thyroid hormones into target cells. However, there have been no reports of avian Oatps to date. The chicken genome project has finally made possible the analysis of avian Oatps, and the present study is the first comprehensive analysis of the avian Oatp/Slco superfamily. Using a database search, we have isolated 10 chicken Oatp genes. Hagenbuch and Meier classified Oatps according to their amino acid sequence identities (3). Oatps within the same family and subfamily share greater than or equal to 40% and greater than or equal to 60% amino acid sequence identities, respectively. Individual families are designated by numerals (e.g. Oatp1, Oatp2, etc.), and individual subfamilies are designated by letters (e.g. Oatp1a, Oatp1b, etc.). In some subfamilies, there are several individual gene products, and in such cases, additional numbering based on the chronology of identification is used (e.g. Oatp1a1, Oatp1a2, etc.). Although these criteria are clear and useful among mammalian species, it is difficult to apply the same criteria to nonmammalian species. Because complete genome information for chicken and other nonmammalian vertebrate species is not yet available, it is currently difficult to assign protein names and gene symbols. However, for convenience, we propose to use temporal nomenclature for avian homologs of the Oatp superfamily as shown in Fig. 1.
Among the 10 Oatps, expression of cOatp1a1, cOatp1b1, cOatp1c1, and cOatp3a2 were observed in the choroid plexus. These Oatps appear to mediate the transmembrane transport of a wide range of endogenous and exogenous amphipathic, organic compounds from the circulating blood to the brain. In the BTH, strong expression of cOatp1c1 and weak expression of cOatp1b1 were observed. Expression of these genes was especially strong in the ependymal cells lining the ventro-lateral walls of the third ventricle. However, no expression was observed in the floor of the third ventricle. Relatively weak expression was also observed around the infundibular nucleus. These regions correlate well with the expression sites of the gene encoding the thyroid hormone inactivating enzyme, type 3 iodothyronine deiodinase (Dio3), and partly correlated with the expression sites of the gene encoding the thyroid hormone activating enzyme, Dio2 (Fig. 4). In quail, the ependymal cells bordering the third ventricle in the BTH are divided into the dorsal, ventro-lateral, and ventral regions; the four types of tanycytes are classified using morphological and metabolic characteristics (17). Type 1 and 4 tanycytes are found in the dorsal ependymal cells and the ventral ependymal cells, respectively. Type 2 and 3 tanycytes are associated with the ventro-lateral ependymal cells. According to the detailed analysis by Sharp (17), tanycytes type 2 and 3 occur in the latero-ventral walls of the BTH dorsal to the posterior median eminence, whereas only type 2 occurs in this region above anterior median eminence. Although cOatp1c1, cOatp1b1, and Dio3 appear to be expressed in the tanycytes in the ventro-lateral region and Dio2 appears to be expressed in the tanycytes in the ventro-lateral and ventral regions, the exact cell that expresses these genes remains unresolved.
Because the signal intensity of cOatp1c1 was approximately 100-fold higher than that of cOatp1b1, and the predominant substrates for mammalian Oatp1c1 and Oatp1b1 are reported to be thyroid hormones and bile salts, respectively, we have focused on cOatp1c1 for further functional analyses. In vitro transport studies using cOatp1c1-expressing CHO cells confirmed that cOatp1c1 transported [125I]T4 with a Km of 6.8 nM and a Vmax of 1.50 pmol per milligram of protein per minute but not [125I]T3. These are consistent with the results obtained in human Oatp1c1 (also known as OATP-F) and rat and mouse Oatp1c1 (also known as Oatp14) (18, 19, 20). Contrary to the fact the rodent counterpart prefers [125I]rT3 as the substrate (18, 19), [125I]rT3 was not transported in avian system. Thus, 3,3',5,5'-iodination is important for efficient transport by cOatp1c1, whereas 3',5'-iodination is required for mammalian Oatp1c1 transport (18, 19). Competition of [125I]T4 uptake with thyroid hormone-related compounds and organic anions that are known to be transported by Oatps also demonstrated the specificity of cOatp1c1. The relative inhibitory activity demonstrated in the present study was consistent with that of mammals although the affinity to the compounds that were tested is higher than those observed in the case of their mammalian counterparts (18, 19, 20). Hence, cOatp1c1 is considered to be a highly specific transporter for T4. We concluded that cOatp1c1 is the avian ortholog of Oatp1c1 (OATP-F in humans or Oatp14 in rodents), and it plays an important role in the delivery of T4 for the generation of the bioactive metabolite T3. The reason for the difference in the substrate specificity between the chicken and mammalian Oatp1c1 remains unknown. Furthermore, although cold T3 significantly inhibited [125I]T4 uptake into the cells that expresses cOatp1c1 and the Ki value of T3 was found to be similar to that of T4, [125I]T3 itself is not a suitable substrate for cOatp1c1. Further studies are required to identify the structural basis for the difference in the substrate preference of Oatps in vertebrates.
The brain is separated from the bloodstream by the blood-brain barrier and the blood-cerebrospinal fluid (CSF) barrier, and the thyroid hormones in the circulating blood have to cross the barriers to exert their effects. cOatp1c1 and transthyretin expressed in the choroid plexus could transport thyroxine into the CSF from the bloodstream (1, 2, 18, 19), whereas cOatp1c1 that was expressed in the ventro-lateral walls of the BTH could play a role in the cellular uptake of thyroxine from the CSF, which is then converted intracellularly either into active T3 by Dio2 or inactive rT3 by Dio3.
In a previous study, we showed that in addition to T3, the level of T4 was also increased in the MBH under LD conditions. However, in the present study, expression levels of Oatps in both the choroid plexus and the BTH show no differences between LD and SD conditions. Although we cannot exclude completely the possibility that the protein level(s) of Oatp(s) or the expression level of an unidentified thyroid transporter may be different between LD conditions and SD conditions, our recent report seems provide an explanation for the increased T4 content under LD conditions (21). Within the BTH, high expression of Dio3 and low expression of Dio2 occurs under SD conditions, whereas under LD conditions, low expression of Dio3 and high expression of Dio2 occurs. Dio3 converts not only T3 but also T4 into inactive metabolites by inner-ring deiodination. Thus, high expression of Dio3 under SD conditions appears to actively eliminate T4 when it is not required (Fig. 9). This reciprocal expression of Dio2 and Dio3 appears to enable birds to fine-tune the thyroid hormone content of the MBH.
In conclusion, we have performed a comprehensive analysis of chicken Oatps. Expression of cOatp1c1 colocalized with Dio2 and Dio3, and cOatp1c1 was shown to transport T4. These results suggest that cOatp1c1 could be involved in the avian photoperiodic response of the gonads. Thyroid hormone plays a critical role in the development and plasticity of the central nervous system, and seasonal morphological changes in the neuro-glial interaction between the GnRH nerve terminals and glial endfeet in the median eminence of quail have been reported (22). To elucidate the molecular mechanism involved in these morphological changes, future studies on the identification of the cellular localization of thyroid hormone receptors and search for the target gene(s) of T3 action are required.
Acknowledgments
We thank Nagoya University Radioisotope Center for use of its facilities.
Footnotes
T.Yo. was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences and a Grant-in-Aid for Encouragement of Young Scientists from the Ministry of Education, Science, Sports and Culture.
First Published Online November 17, 2005
1 The first four authors contributed equally to this work.
Abbreviations: BTH, Basal tuberal hypothalamus; CHO, Chinese hamster ovary; CSF, cerebrospinal fluid; Dio2, type 2 iodothyronine deiodinase; Dio3, type 3 iodothyronine deiodinase; LD, long day; MBH, mediobasal hypothalamus; Oatp, organic anion transporting polypeptide; SD, short day.
Accepted for publication November 10, 2005.
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