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• W1979697072 abstract "Retinoic acid (RA) and thyroid hormone are critical for differentiation and organogenesis in the embryo. Mct8 (monocarboxylate transporter 8), expressed predominantly in the brain and placenta, mediates thyroid hormone uptake from the circulation and is required for normal neural development. RA induces differentiation of F9 mouse teratocarcinoma cells toward neurons as well as extraembryonal endoderm. We hypothesized that Mct8 is functionally expressed in F9 cells and induced by RA. All-trans-RA (tRA) and other RA receptor (RAR) agonists dramatically (>300-fold) induced Mct8. tRA treatment significantly increased uptake of triiodothyronine and thyroxine (4.1- and 4.3-fold, respectively), which was abolished by a selective Mct8 inhibitor, bromosulfophthalein. Sequence inspection of the Mct8 promoter region and 5′-rapid amplification of cDNA ends PCR analysis in F9 cells identified 11 transcription start sites and a proximal Sp1 site but no TATA box. tRA significantly enhanced Mct8 promoter activity through a consensus RA-responsive element located 6.6 kilobases upstream of the coding region. A chromatin immunoprecipitation assay demonstrated binding of RAR and retinoid X receptor to the RA response element. The promotion of thyroid hormone uptake through the transcriptional up-regulation of Mct8 by RAR is likely to be important for extraembryonic endoderm development and neural differentiation. This finding demonstrates cross-talk between RA signaling and thyroid hormone signaling in early development at the level of the thyroid hormone transporter. Retinoic acid (RA) and thyroid hormone are critical for differentiation and organogenesis in the embryo. Mct8 (monocarboxylate transporter 8), expressed predominantly in the brain and placenta, mediates thyroid hormone uptake from the circulation and is required for normal neural development. RA induces differentiation of F9 mouse teratocarcinoma cells toward neurons as well as extraembryonal endoderm. We hypothesized that Mct8 is functionally expressed in F9 cells and induced by RA. All-trans-RA (tRA) and other RA receptor (RAR) agonists dramatically (>300-fold) induced Mct8. tRA treatment significantly increased uptake of triiodothyronine and thyroxine (4.1- and 4.3-fold, respectively), which was abolished by a selective Mct8 inhibitor, bromosulfophthalein. Sequence inspection of the Mct8 promoter region and 5′-rapid amplification of cDNA ends PCR analysis in F9 cells identified 11 transcription start sites and a proximal Sp1 site but no TATA box. tRA significantly enhanced Mct8 promoter activity through a consensus RA-responsive element located 6.6 kilobases upstream of the coding region. A chromatin immunoprecipitation assay demonstrated binding of RAR and retinoid X receptor to the RA response element. The promotion of thyroid hormone uptake through the transcriptional up-regulation of Mct8 by RAR is likely to be important for extraembryonic endoderm development and neural differentiation. This finding demonstrates cross-talk between RA signaling and thyroid hormone signaling in early development at the level of the thyroid hormone transporter. Retinoic acid (RA) and thyroid hormones are essential for vertebrate development (1Mark M. Ghyselinck N.B. Chambon P. Nucl. Recept. Signal. 2009; 7: e002Crossref PubMed Scopus (279) Google Scholar, 2Yen P.M. Physiol. Rev. 2001; 81: 1097-1142Crossref PubMed Scopus (1542) Google Scholar). The actions of these hormones are mediated by specific nuclear hormone receptors, RA receptor (RAR) 3The abbreviations used are: RARretinoic acid receptorBCH2-amino-2-norbornane carboxylic acidBSPbromosulfophthaleinDBTSSdatabase of transcriptional start sitesDRdirect repeatLatL-type amino acid transporterLucluciferaseRACErapid amplification of cDNA endsRAREretinoic acid response elementRXRretinoid X receptorT3triiodothyronineT4thyroxineTRthyroid hormone receptortRAall-trans-retinoic acidTSStranscription start site. and thyroid hormone receptor (TR), respectively. RAR signaling plays a critical role in embryonic patterning and in organogenesis (1Mark M. Ghyselinck N.B. Chambon P. Nucl. Recept. Signal. 2009; 7: e002Crossref PubMed Scopus (279) Google Scholar). TR modulates RA-stimulated neural differentiation as well as expression of some RA-responsive genes in embryonic stem cells (3Lee L.R. Mortensen R.M. Larson C.A. Brent G.A. Mol. Endocrinol. 1994; 8: 746-756PubMed Google Scholar, 4Liu Y.Y. Tachiki K.H. Brent G.A. Endocrinology. 2002; 143: 2664-2672Crossref PubMed Google Scholar). The timing of ligand availability and receptor expression is important for normal neural differentiation (4Liu Y.Y. Tachiki K.H. Brent G.A. Endocrinology. 2002; 143: 2664-2672Crossref PubMed Google Scholar). retinoic acid receptor 2-amino-2-norbornane carboxylic acid bromosulfophthalein database of transcriptional start sites direct repeat L-type amino acid transporter luciferase rapid amplification of cDNA ends retinoic acid response element retinoid X receptor triiodothyronine thyroxine thyroid hormone receptor all-trans-retinoic acid transcription start site. RAR and TR form heterodimers with retinoid X (9-cis-RA) receptor (RXR) for regulation of most target genes. The consensus sequence of an RA response element (RARE) contains a direct repeat of the consensus half-sites, 5′-PuG(G/T)(T/A)CA-3′, with spacing of 1, 2, or 5 bases (DR-1, DR-2, or DR-5), whereas that of a retinoid X (or 9-cis-RA) response element contains the same half-sites separated by 1 base (DR-1) (5Giguère V. Endocr. Rev. 1994; 15: 61-79Crossref PubMed Google Scholar). Although the difference of half-site spacing provides selectivity for a specific receptor, there are interactions among the various nuclear receptor signaling pathways. Nuclear receptors also share common co-activator(s) and co-repressor(s) for transcriptional regulation (2Yen P.M. Physiol. Rev. 2001; 81: 1097-1142Crossref PubMed Scopus (1542) Google Scholar). A shared requirement for RXR, interaction at related cis-elements, and competition for co-factors are some of the mechanisms underlying cross-talk among nuclear receptor signaling pathways in development (6Furlow J.D. Neff E.S. Trends Endocrinol. Metab. 2006; 17: 40-47Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) and in metabolic regulation (2Yen P.M. Physiol. Rev. 2001; 81: 1097-1142Crossref PubMed Scopus (1542) Google Scholar). Nuclear receptor ligands are generally lipophilic and have been thought to reach their receptor by passive diffusion through the plasma membrane. Recent studies, however, have demonstrated that uptake of some of these ligands is mediated by selective transporters. Several members of the solute carrier (Slc) family (7Jansen J. Friesema E.C. Milici C. Visser T.J. Thyroid. 2005; 15: 757-768Crossref PubMed Scopus (132) Google Scholar, 8Friesema E.C. Jansen J. Jachtenberg J.W. Visser W.E. Kester M.H. Visser T.J. Mol. Endocrinol. 2008; 22: 1357-1369Crossref PubMed Scopus (213) Google Scholar, 9van der Deure W.M. Peeters R.P. Visser T.J. Best Pract. Res. Clin. Endocrinol. Metab. 2007; 21: 339-350Crossref PubMed Scopus (22) Google Scholar), including Mct8 (monocarboxylate transporter-8, or Slc16a2) (10Friesema E.C. Ganguly S. Abdalla A. Manning Fox J.E. Halestrap A.P. Visser T.J. J. Biol. Chem. 2003; 278: 40128-40135Abstract Full Text Full Text PDF PubMed Scopus (546) Google Scholar), and the solute carrier organic anion transporter family (Slco, or organic anion-transporting polypeptides, Oatp) (9van der Deure W.M. Peeters R.P. Visser T.J. Best Pract. Res. Clin. Endocrinol. Metab. 2007; 21: 339-350Crossref PubMed Scopus (22) Google Scholar, 11Hagenbuch B. Best Pract. Res. Clin. Endocrinol. Metab. 2007; 21: 209-221Crossref PubMed Scopus (104) Google Scholar) are known as thyroid hormone transporters. Mct8 loss of function mutations in humans are associated with profound neurological deficits (7Jansen J. Friesema E.C. Milici C. Visser T.J. Thyroid. 2005; 15: 757-768Crossref PubMed Scopus (132) Google Scholar), a common manifestation of thyroid hormone insufficiency in embryos and fetuses. Mct8 is expressed in many tissues, including brain, placenta, liver, and kidney (7Jansen J. Friesema E.C. Milici C. Visser T.J. Thyroid. 2005; 15: 757-768Crossref PubMed Scopus (132) Google Scholar, 10Friesema E.C. Ganguly S. Abdalla A. Manning Fox J.E. Halestrap A.P. Visser T.J. J. Biol. Chem. 2003; 278: 40128-40135Abstract Full Text Full Text PDF PubMed Scopus (546) Google Scholar, 12Chan S.Y. Franklyn J.A. Pemberton H.N. Bulmer J.N. Visser T.J. McCabe C.J. Kilby M.D. J. Endocrinol. 2006; 189: 465-471Crossref PubMed Scopus (70) Google Scholar), which are all important thyroid hormone targets. F9 teratocarcinoma cells have been widely used as an in vitro model of embryonic stem cell differentiation. A combination treatment of F9 cells with all-trans-RA (tRA), an agonist of RAR, and cAMP induces extraembryonic endoderm (13Soprano D.R. Teets B.W. Soprano K.J. Vitam. Horm. 2007; 75: 69-95Crossref PubMed Scopus (134) Google Scholar, 14Komiya S. Shimizu M. Ikenouchi J. Yonemura S. Matsui T. Fukunaga Y. Liu H. Endo F. Tsukita S. Nagafuchi A. Genes Cells. 2005; 10: 1065-1080Crossref PubMed Scopus (19) Google Scholar) or neuron-like cells (15Kuff E.L. Fewell J.W. Dev. Biol. 1980; 77: 103-115Crossref PubMed Scopus (66) Google Scholar), depending on the composition of culture media. The extraembryonic endoderm supports the developing embryo and facilitates exchange of small molecules between the maternal circulation and the embryo, functioning as an “early placenta” (16Cross J.C. Werb Z. Fisher S.J. Science. 1994; 266: 1508-1518Crossref PubMed Scopus (1188) Google Scholar). Because mature placenta expresses abundant Mct8 and transports thyroid hormone (12Chan S.Y. Franklyn J.A. Pemberton H.N. Bulmer J.N. Visser T.J. McCabe C.J. Kilby M.D. J. Endocrinol. 2006; 189: 465-471Crossref PubMed Scopus (70) Google Scholar), it may be expressed in F9 cells differentiated into extraembryonic endoderm. Thyroid hormone is critical for brain development, and thus neural differentiation in F9 cells should be accompanied by induction of thyroid hormone transporter(s). To test these hypotheses, we investigated effects of the differentiation inducer, tRA, on expression of Mct8 and other thyroid hormone transporter genes as well as thyroid hormone uptake in F9 cells. F9 cells, purchased from ATCC (Manassas, VA), were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) (Invitrogen) in gelatin-coated flasks or Petri dishes as recommended. Cells were subcultivated at a ratio of 1:10 to 1:20 and used at passages 2–6, unless otherwise noted. Neuron-like cells were induced as described previously (15Kuff E.L. Fewell J.W. Dev. Biol. 1980; 77: 103-115Crossref PubMed Scopus (66) Google Scholar) with minor modifications. Briefly, ∼5 × 105 cells seeded in gelatin-coated Petri dishes were maintained in DMEM/F-12 medium (Invitrogen) with 2% FBS, 1 μm tRA, and 1 mm 8-bromo-cAMP for up to 7 days. The cells were fed every 2 days with fresh medium. The cells were split again at a subcultivation ratio of 1:10 2 days after the first seeding. JEG3 cells, MCF7 cells, and SH-SY5Y cells were purchased from ATCC and maintained as recommended. Synthetic retinoids were synthesized as described (17Kagechika H. Kawachi E. Hashimoto Y. Himi T. Shudo K. J. Med. Chem. 1988; 31: 2182-2192Crossref PubMed Scopus (256) Google Scholar, 18Kagechika H. Kawachi E. Hashimoto Y. Shudo K. J. Med. Chem. 1989; 32: 834-840Crossref PubMed Scopus (80) Google Scholar), dissolved in dimethyl sulfoxide to 10−2 m, and stored at −20 °C. Restriction enzymes and DNA modification enzymes were purchased from New England Biolabs (Ipswich, MA). Other chemicals were purchased from Sigma unless otherwise noted. Two-step quantitative RT-PCR was carried out by using the DNA Engene Opticon System (MJ Research, Waltham, MA) as described (19Kogai T. Kanamoto Y. Li A.I. Che L.H. Ohashi E. Taki K. Chandraratna R.A. Saito T. Brent G.A. Endocrinology. 2005; 146: 3059-3069Crossref PubMed Scopus (47) Google Scholar) with minor modifications. Briefly, total RNA from culture cells was prepared with the RNeasy minikit (Qiagen, Valencia, CA) with on-column DNase digestion. Three μg of total RNA was reverse-transcribed by using 50 units of Superscript III reverse transcriptase (Invitrogen) in a 20-μl reaction with oligo(dT)12–18 primer or random hexamer. Quantitative PCR of mouse thyroid hormone transporter genes, glyceraldehyde-3-phosphate dehydrogenase gene (Gapdh), 18 S ribosomal RNA, and human MCT8 mRNA was performed with custom DNA primers synthesized by Invitrogen (supplemental Table 1). Quantitative PCR of markers of extraembryonic endoderm and neural differentiation was carried out with the QuantiTect primer assay (Qiagen). Standard curves representing 6-point serial dilution of the corresponding control group were analyzed in each assay and used for calculation of relative expression values. RT-PCR of human GAPDH was performed as previously described (19Kogai T. Kanamoto Y. Li A.I. Che L.H. Ohashi E. Taki K. Chandraratna R.A. Saito T. Brent G.A. Endocrinology. 2005; 146: 3059-3069Crossref PubMed Scopus (47) Google Scholar). The sample quantifications were normalized by the internal control Gapdh or 18 S RNA. Conventional two-step RT-PCR of the 5′-untranslated region of Mct8 was performed with custom primers (supplemental Table 1) by using the Expand High Fidelity PCR system (Roche Applied Science). The cycle number was 35. Uptake of triiodothyronine (T3) and thyroxine (T4) was measured as described previously (20Friesema E.C. Kuiper G.G. Jansen J. Visser T.J. Kester M.H. Mol. Endocrinol. 2006; 20: 2761-2772Crossref PubMed Scopus (169) Google Scholar) with minor modifications. Cells, grown in 12-well plates, as well as empty wells for measurement of nonspecific binding of radiolabeled thyroid hormone to the surface of the side wall of the well were rinsed with 1 ml of Dulbecco's PBS, preincubated with 300 μl of Hanks' balanced salt solution with 0.1% bovine serum albumin (BSA) for 15 min at 37 °C, and the medium was replaced with 300 μl of preheated thyroid hormone uptake assay buffer. T3 uptake assay buffer contains Hanks' balanced salt solution, 0.1% BSA, 0.25 μCi/ml 125I-labeled T3 (MP Biomedicals, Solon, OH), and 1.0 nm T3. T4 uptake assay buffer contains Hanks' balanced salt solution, 0.1% BSA, 0.3 μCi/ml 125I-labeled T4 (MP Biomedicals), and 1.0 μm T4. Cells were incubated for 4–30 min at 37 °C in a humidified incubator, rinsed twice with 1 ml of ice-cold Dulbecco's PBS, and lysed with 200 μm passive lysis buffer (Promega). Radioactivity of the whole lysate as well as lysis buffer from the duplicate empty wells was measured in a γ-counter. The background count from the side wall was subtracted from the count of cell lysate. The count was then normalized to the cellular protein content measured in the same cells by using a Bio-Rad protein assay. To determine putative RARE, consensus half-sites, (A/G)G(G/T)(A/T)CA as well as other reported half-sites (5Giguère V. Endocr. Rev. 1994; 15: 61-79Crossref PubMed Google Scholar) were searched on both strands of the mouse Mct8 locus (NT_000086) by using MacMolly Tetra Lite (Mologen, Berlin, Germany) as described (21Kogai T. Ohashi E. Jacobs M.S. Sajid-Crockett S. Fisher M.L. Kanamoto Y. Brent G.A. J. Clin. Endocrinol. Metab. 2008; 93: 1884-1892Crossref PubMed Scopus (28) Google Scholar). CpG islands around the transcription start site (TSS) of Mct8 were predicted by the CpG island searcher (hosted on the worldwide web by the laboratory of Dr. P. A. Jones at University of Southern California (USC)) with the following parameters: observed/expected ratio, >0.65; %GC, >50; length, >200 (22Gardiner-Garden M. Frommer M. J. Mol. Biol. 1987; 196: 261-282Crossref PubMed Scopus (2661) Google Scholar). Basic transcription factor binding sites (23Sandelin A. Carninci P. Lenhard B. Ponjavic J. Hayashizaki Y. Hume D.A. Nat. Rev. Genet. 2007; 8: 424-436Crossref PubMed Scopus (398) Google Scholar, 24Frith M.C. Valen E. Krogh A. Hayashizaki Y. Carninci P. Sandelin A. Genome Res. 2008; 18: 1-12Crossref PubMed Scopus (183) Google Scholar) were searched around the TSS of Mct8 by using MacMolly Tetra Lite. Total RNA from F9 cells treated with 1 μm tRA for 72 h was isolated with the RNeasy Plus kit (Qiagen). The 5′-end of the Mct8 cDNA was identified by the oligo-capping and RNA ligase-mediated RACE method with the GeneRacer kit (Invitrogen) according to the manufacturer's instructions. Briefly, 5 μg of total RNA was dephosphorylated, decapped, and ligated to GeneRacer RNA oligonucleotides. Reverse transcription was carried out with random primer by using SuperScript III reverse transcriptase (Invitrogen). The 5′-ends of Mct8 were amplified from the cDNA pool as a template with the GeneRacer 5′-primer and +496 primer (supplemental Table 1). Nested PCR was then performed with the GeneRacer 5′-nested primer and the +496 primer. PCR products were cloned into pCR4-TOPO (Invitrogen) and analyzed by automated DNA sequencer (Laguna Scientific Laboratory, Laguna Niguel, CA). To generate constructs for screening of functional RARE on mouse Mct8, annealed synthetic oligonucleotides, containing the putative RAREs as well as an exogenous BamHI site for confirmation of cloning (supplemental Table 1), were inserted to the SmaI site of the pGL3 promoter vector (Promega, Madison, WI). A DNA fragment of the Mct8 5′-flanking region (−976 to −54; +1 is A in the translation start site) was obtained by genomic PCR from F9 cells with a forward primer (5′-CAGATCTTTCGTGCCTCCCTCCTTTC-3′) and a reverse primer (5′-CAAGCTTGTTTCTGCTGCTACTGCTCCT-3′). To generate pGL3 −976/−54, the PCR products were ligated into the polylinker site of the pGL Basic vector (Promega) with BglII and HindIII. pGL3 −836/−54 and pGL3 −659/−54 were constructed from the pGL3 −976/−54 by deletion of the sequences between ApaI and BglII sites and AgeI and BglII sites, respectively, both of which were treated with T4 DNA polymerase. pGL3 DR5A −976/−54 and pGL3 DR5A −836/−54 were constructed by blunt-end ligation of annealed oligonucleotides of the DR5A (supplemental Table 1) into the SmaI site of the pGL3 −976/−54 and the pGL3 −836/−54, respectively. Mutation of the DR5A element in pGL3 DR5A −836/−54 was generated by using the QuikChange Lightning site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA) with custom primers from Invitrogen (supplemental Table 1). Sequences of the constructed vectors were analyzed by an automated DNA sequencer (Laguna Scientific Laboratory). Cells (500–600 cells/well) were seeded in 12-well dishes 24 h before transfection. Unless otherwise noted, 0.2 μg of a Luciferase (Luc) reporter construct and 10 ng of a Renilla Luc reporter vector, pRL-CMV (Promega), were transfected to the cells by using Effectene (Qiagen, Valencia, CA) as recommended. The transfection medium was changed to growth media with or without RA at 24 h, and the Luc assay was performed at 48 h with a dual Luc reporter assay system (Promega). Results of the Luc reporter assay were normalized to Renilla Luc expression. F9 cells, grown in 100-mm Petri dishes, were treated with tRA (1 μm) for up to 90 min and fixed with 1% formaldehyde. The ChIP assay was carried out with the ChIP-IT express enzymatic kit (Active Motif, Carlsbad, CA) as recommended. Partially digested chromatin was immunoprecipitated with 3 μg of anti-RARα (C-20), anti-RARβ (C-19), or anti-RXRα (D-20) antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) overnight at 4 °C. Eluted DNA, as well as the aliquot of sheared chromatin prior to immunoprecipitation (input), was amplified by using the Expand high fidelity PCR system (Roche Applied Science) with primers specific to an RA-responsive region of mouse Mct8 or the mouse Lat1 (L-type amino acid transporter 1) promoter (supplemental Table 1). PCR cycle numbers for Mct8 and Lat1 were 40 and 37, respectively. The amplicons were analyzed by electrophoresis in a 2% agarose gel. tRA treatment markedly increased Mct8 mRNA expression in F9 cells grown in media with 10% serum. A time course study (Fig. 1A) showed a significant induction of Mct8 mRNA by tRA (1 μm) at 12 h (∼9.5-fold), reaching a maximum at 96 h (∼1120-fold). The effect was dose-dependent with a 50% effective tRA concentration (EC50) of ∼1.47 × 10−7 m and maximum induction at an RA concentration of 10−6 m (Fig. 1B). In the presence of 10% charcoal-stripped FBS, the EC50 for tRA was ∼1.35 × 10−7 m. tRA is a potent agonist of RAR, although some tRA is converted intracellularly to 9-cis-RA, an agonist of RXR. Undifferentiated F9 cells express both RAR and RXR. To determine which receptor is required for Mct8 induction, we utilized synthetic retinoid receptor agonists (17Kagechika H. Kawachi E. Hashimoto Y. Himi T. Shudo K. J. Med. Chem. 1988; 31: 2182-2192Crossref PubMed Scopus (256) Google Scholar, 18Kagechika H. Kawachi E. Hashimoto Y. Shudo K. J. Med. Chem. 1989; 32: 834-840Crossref PubMed Scopus (80) Google Scholar). Treatment with the pan-RAR agonist, Re80 (1 μm), or with an RARα/β-specific agonist, Am80 (1 μm), mimicked the effects of tRA on Mct8 mRNA expression. No significant induction was observed with the pan-RXR agonists, HX630 and PA024 (Fig. 1C). The induction of Mct8 by tRA was significantly inhibited by an RAR antagonist, LE135, as well as RXR antagonists HX531 and PA452 (Fig. 1D). These data indicate that stimulation of RAR is required for the induction of Mct8 in F9 cells. Expression of RXR, but not stimulation by ligand, is also probably required for the up-regulation of Mct8. Both RAR and RXR are expressed in many types of cancer cells, including MCF-7 breast cancer cells (25Kogai T. Kanamoto Y. Che L.H. Taki K. Moatamed F. Schultz J.J. Brent G.A. Cancer Res. 2004; 64: 415-422Crossref PubMed Scopus (56) Google Scholar) and SH-SY5Y neuroblastoma cells. Our quantitative RT-PCR study indicated abundant Mct8 expression in untreated SH-SY5Y cells (∼36% of the level in tRA-treated F9 cells) (Table 1). tRA, however, did not significantly increase the MCT8 mRNA expression in SH-SY5Y or MCF7 cells (Table 1). JEG3 cells express abundant RXR but not RAR, and treatment with tRA did not induce MCT8 (Table 1).TABLE 1Expression of MCT8 mRNA in various cancer cell lines−tRA+tRA+tRA/−tRAF91.00 ± 0.0877.4 ± 4.46ap < 0.01, when compared with untreated cells.76.9 ± 8.32JEG32.03 × 10−4 ± 3.27 × 10−51.41 × 10−4 ± 8.50 × 10−50.74 ± 0.05MCF70.30 ± 0.010.51 ± 0.041.71 ± 0.19SH-SY5Y27.8 ± 3.3420.5 ± 3.540.73 ± 0.04a p < 0.01, when compared with untreated cells. Open table in a new tab Transfection of Mct8 into mammalian cells induces uptake of both T3 and T4 (20Friesema E.C. Kuiper G.G. Jansen J. Visser T.J. Kester M.H. Mol. Endocrinol. 2006; 20: 2761-2772Crossref PubMed Scopus (169) Google Scholar). To investigate if the induction of endogenous Mct8 by tRA increases functional Mct8 expression, we measured the accumulation of T3 as well as T4 in F9 cells treated with or without tRA (1 μm) for 6 days. Cells were incubated with 125I-labeled T3 or T4 for 4–30 min in the presence of 0.1% BSA as a carrier. T3 uptake in tRA-treated cells was significantly increased in the first 4 min and partially saturated at 10 min (Fig. 2A). T3 uptake in tRA-treated cells at 10 min was significantly higher (∼4.1-fold) than that in untreated cells (Fig. 2A). T4 uptake was also significantly increased in the first 4 min and saturated at 10 min (Fig. 2B). T4 uptake in tRA-treated cells at 10 min was significantly higher (∼4.3-fold) than that in untreated cells (Fig. 2B). These results are consistent with previous data of thyroid hormone uptake in mammalian cells transfected with vectors expressing Mct8 (20Friesema E.C. Kuiper G.G. Jansen J. Visser T.J. Kester M.H. Mol. Endocrinol. 2006; 20: 2761-2772Crossref PubMed Scopus (169) Google Scholar). Although Mct8 is one of the most efficient transporters of both T3 and T4, several other transporters have also been reported to mediate thyroid hormone uptake, including other SLC family members, Mct10 (or SLC16A10) (8Friesema E.C. Jansen J. Jachtenberg J.W. Visser W.E. Kester M.H. Visser T.J. Mol. Endocrinol. 2008; 22: 1357-1369Crossref PubMed Scopus (213) Google Scholar), sodium/taurocholate-cotransporting polypeptide-1 (NTCP1, or SLC10A1), L-type amino acid transporter-1 (LAT1, or SLC7A5) and LAT2 (or SLC7A8) (9van der Deure W.M. Peeters R.P. Visser T.J. Best Pract. Res. Clin. Endocrinol. Metab. 2007; 21: 339-350Crossref PubMed Scopus (22) Google Scholar), and eight of the 15 members of the Oatp/Slco family (9van der Deure W.M. Peeters R.P. Visser T.J. Best Pract. Res. Clin. Endocrinol. Metab. 2007; 21: 339-350Crossref PubMed Scopus (22) Google Scholar, 11Hagenbuch B. Best Pract. Res. Clin. Endocrinol. Metab. 2007; 21: 209-221Crossref PubMed Scopus (104) Google Scholar). To determine if the tRA-induced thyroid hormone uptake in F9 cells is mediated by Mct8, we utilized pharmacological inhibitors of thyroid hormone transporters (26Wirth E.K. Roth S. Blechschmidt C. Hölter S.M. Becker L. Racz I. Zimmer A. Klopstock T. Gailus-Durner V. Fuchs H. Wurst W. Naumann T. Bräuer A. de Angelis M.H. Köhrle J. Grüters A. Schweizer U. J. Neurosci. 2009; 29: 9439-9449Crossref PubMed Scopus (147) Google Scholar). An inhibitor of T3 uptake by Mct8, bromosulfophthalein (BSP), but not a broad spectrum inhibitor of Oatp, probenecid, significantly reduced the tRA-induced T3 uptake (Fig. 2C) with an IC50 of 112 μm (Fig. 2D). Modest T3 uptake was observed in the cells without tRA treatment, although it was not inhibited by BSP or probenecid (Fig. 2A). A Lat inhibitor, 2-amino-2-norbornane carboxylic acid (BCH), significantly reduced the T3 uptake in both tRA-treated and -untreated F9 cells, whereas the induction by tRA was not abolished (Fig. 2C). These data suggest that the basal modest T3 uptake is mediated by Lat, whereas tRA-induced T3 uptake is dependent on the BSP-sensitive thyroid hormone transporter, Mct8. To determine if the tRA-induced thyroid hormone uptake (Fig. 2) was due to induction of Mct8, we measured the expression of mouse orthologs of other reported thyroid hormone transporter genes in response to tRA treatment in F9 cells (9van der Deure W.M. Peeters R.P. Visser T.J. Best Pract. Res. Clin. Endocrinol. Metab. 2007; 21: 339-350Crossref PubMed Scopus (22) Google Scholar, 11Hagenbuch B. Best Pract. Res. Clin. Endocrinol. Metab. 2007; 21: 209-221Crossref PubMed Scopus (104) Google Scholar). The expression level of each gene was quantified by RT-PCR with a Gapdh standard for comparison of the relative expression among each gene. tRA treatment for 96 h increased expression of Mct10 and Lat2 (Table 2), although the magnitudes of induction (∼2.1- and ∼4.6-fold, respectively) were much less than that of Mct8 (∼678-fold). Interestingly, abundant expression of Lat1 (∼7 × 10−2-fold compared with Gapdh), as well as modest expression of Oatp4a1 (∼10−3-fold compared with Gapdh), was observed in both tRA-treated and untreated F9 cells, although tRA did not significantly influence the expression of these transporters (Table 2). Expression levels of Ntcp1 and the other seven Oatp genes were relatively small (less than 5 × 10−5-fold compared with Gapdh) and were not significantly increased by the tRA treatment (Table 2). These data indicate that, among the thyroid hormone transporter genes tested in F9 cells, only Mct8 was markedly induced by tRA.TABLE 2Expression of thyroid hormone transporter genes in F9 cells−tRA+tRA+tRA/−tRAMct8 (Slc16a2)1.92 × 10−5 ± 1.29 × 10−61.29 × 10−2 ± 1.25 × 10−3ap < 0.01, when compared with −tRA.678.1 ± 78.6Mct10 (Slc16a10)1.51 × 10−4 ± 3.73 × 10−53.11 × 10−4 ± 2.78 × 10−5ap < 0.01, when compared with −tRA.2.05 ± 0.53Ntcp1 (Slc10a1)<10−6<10−6NALat1 (Slc7a5)7.26 × 10−2 ± 2.11 × 10−36.68 × 10−2 ± 2.08 × 10−30.96 ± 0.18Lat2 (Slc7a8)8.83 × 10−5 ± 5.51 × 10−64.08 × 10−4 ± 5.49 × 10-6ap < 0.01, when compared with −tRA.4.63 ± 0.21Oatp (Slco) 1a14.66 × 10−5 ± 5.23 × 10−73.37 × 10−5 ± 3.21 × 10−60.72 ± 0.05Oatp (Slco) 1a4<10−6<10−6NAOatp (Slco) 1a5<10−6<10−6NAOatp (Slco) 1b21.10 × 10−5 ± 6.45 × 10−75.20 × 10−6 ± 4.26 × 10−70.47 ± 0.05Oatp (Slco) 1c1<10−6<10−6NAOatp (Slco) 4a11.45 × 10−3 ± 9.48 × 10−52.35 × 10−4 ± 2.15 × 10−50.16 ± 0.002Oatp (Slco) 6b1<10−6<10−6NAOatp (Slco) 6c1<10−6<10−6NAGapdh1.29 ± 0.020.93 ± 0.140.72 ± 0.07a p < 0.01, when compared with −tRA. Open table in a new tab Treatment of F9 cells with tRA and/or cAMP under normal (10%) serum conditions induces extraembryonic endoderm, primitive, parietal, and visceral (13Soprano D.R. Teets B.W. Soprano K.J. Vitam. Horm. 2007; 75: 69-95Crossref PubMed Scopus (134) Google Scholar, 14Komiya S. Shimizu M. Ikenouchi J. Yonemura S. Matsui T. Fukunaga Y. Liu H. Endo F. Tsukita S. Nagafuchi A. Genes Cells. 2005; 10: 1065-1080Crossref PubMed Scopus (19) Google Scholar). Combination treatment with tRA and cAMP, under low serum conditions, induces morphologically neuron-like cells with neuron-specific acetylcholinesterase activity (15Kuff E.L. Fewell J.W. Dev. Biol. 1980; 77: 103-115Crossref PubMed Scopus (66) Google Scholar). tRA treatment induced primitive/visceral endoderm differentiation markers, Col4a1, Lama1, and Afp, in F9 cells, whereas the combination with tRA and 8-bromo-cAMP induced parietal endoderm differentiation markers, Col4a1, Lama1, and Thbd (Fig. 3, A–C), consistent with previous reports (13Soprano D.R. Teets B.W. Soprano K.J. Vitam. Horm. 2007; 75: 69-95Crossref PubMed Scopus (134) Google Scholar, 14Komiya S. Shimizu M. Ikenouchi J. Yonemura S. Matsui T. Fukunaga Y. Liu H. Endo F. Tsukita S. Nagafuchi A. Genes Cells. 2005; 10: 1065-1080Crossref PubMed Scopus (19) Google Scholar). The expression of Mc" @default.
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• W1979697072 title "Retinoic Acid Induces Expression of the Thyroid Hormone Transporter, Monocarboxylate Transporter 8 (Mct8)" @default.
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