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Follicular Thyroglobulin Enhances Gene Expression Necessary for Thyroid Hormone Secretion

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Follicular Thyroglobulin Enhances Gene Expression Necessary for Thyroid Hormone Secretion Endocrine Journal 2015, 62 (11), 1007-1015 Original Follicular thyroglobulin enhances gene expression necessary for thyroid hormone secretion Yuko Ishido1), 2), 3), Yuqian Luo1), 3), Aya Yoshihara1), 3), Moyuru Hayashi3), Akio Yoshida2), Ichiro Hisatome2) and Koichi Suzuki1), 3) 1) Laboratory of Molecular Diagnostics, Department of Mycobacteriology, Leprosy Research Center, National Institute of Infectious Disease, Tokyo 189-0002, Japan 2) Division of Regenerative Medicine and Therapeutics, Department of Genetic Medicine and Regenerative Therapeutics, Institute of Regenerative Medicine and Biofunction, Tottori University Graduate School of Medical Science, Yonago, 683-8503, Japan 3) Department of Clinical Laboratory Science, Faculty of Medical Technology, Teikyo University, Tokyo 173-8605, Japan Abstract. We have previously shown that follicular thyroglobulin (Tg) has an unexpected function as an autocrine negative- feedback regulator of thyroid hormone (TH) biosynthesis. Tg significantly suppressed the expression of genes necessary for iodide transport and TH synthesis by counteracting stimulation by TSH. However, whether follicular Tg also regulates intracellular TH transport and its secretion from thyrocytes is not known. In the present study, we examined the potential effect of follicular Tg on TH transport and secretion by quantifying the expression of two TH transporters: monocarboxylate transporter 8 (MCT8) and μ-crystallin (CRYM). Our results showed that follicular Tg at physiologic concentrations enhanced both the mRNA and protein expression levels of MCT8 and CRYM in a time- and dose-dependent manner in rat thyroid FRTL-5 cells. Although both the sodium/iodide symporter (NIS), an essential transporter of iodide from blood into the thyroid, and MCT8, a transporter of synthesized TH from the gland, were co-localized on the basolateral membrane of rat thyrocytes in vivo, Tg decreased NIS expression and increased the expression of MCT8 by counteracting TSH action. Thus, the effect of Tg on TH secretion opposed its previously described negative-feedback suppression of TH synthesis. Our results indicate that Tg mediates a complex intrinsic regulation of gene expression that is necessary to balance two opposing vectorial transport systems: the inflow of newly synthesized TH and the outflow of TH by external secretion. Key words: Thyroglobulin, Thyroid hormone, MCT8, CRYM THYROGLOBULIN (Tg), the most abundant product receptor (Tshr), thyroid transcription factor 1 (TTF1; synthesized by thyrocytes and stored in the thyroid fol- Nkx2–1), thyroid transcription factor 2 (TTF2; Foxe1), licles, serves as the macromolecular protein backbone and paired box gene 8 (Pax8), all of which are essen- for thyroid hormone (TH) biosynthesis through cova- tial participants in the ‘assembly line’ of TH produc- lent incorporation of iodide into its tyrosyl residues [1]. tion [2-6]. Indeed, Tg inhibited TSH-stimulated iodine In addition to this well recognized function, Tg stored uptake and H2O2 generation, which are essential for in the follicle has been shown to have an unexpected TH synthesis [2, 5, 6]. Such negative-feedback mech- role as a negative-feedback regulator of TH synthesis anisms have also been observed in primary cultures of [2-7]. Thus, in rat thyroid FRTL-5 cells and rat thy- normal human thyrocytes [7]. Based on these stud- roid glands in vivo, follicular Tg suppressed the expres- ies, it has become apparent that the homeostasis of TH sion of Tg, the sodium/iodide symporter (NIS; Slc5a5), synthesis is physiologically balanced by TSH-induced thyroid peroxidase (Tpo), dual oxidase 2 (Duox2), dual stimulation from the basal side of the thyroid follicular oxidase maturation factor 2 (Duoxa2), the thyrotropin cells and the negative-feedback action of follicular Tg from the apical side. Submitted May 2, 2015; Accepted Aug. 19, 2015 as EJ15-0263 The question of whether Tg affects the secretion of Released online in J-STAGE as advance publication Sep. 11, 2015 TH from thyrocytes has not been investigated to date. Correspondence to: Koichi Suzuki, Department of Clinical Laboratory Science, Faculty of Medical Technology, Teikyo In contrast to the iodide uptake and TH synthesis pro- University, 2-11-1 Kaga, Itabashi, Tokyo 173-8605, Japan. cesses, the mechanisms involved in the transport and E-mail: [email protected] secretion of synthesized TH in thyrocytes are not well ©The Japan Endocrine Society 1008 Ishido et al. understood, although TSH is known to stimulate these Crym reverse, 5′-CTACTGGCTCCAACAGCATTGA-3′; processes [8, 9]. Recently, energy-dependent trans- Gapdh forward, 5′-ACAGCAACAGGGTGGTGGAC-3′; porters that mediate TH secretion have been identi- and Gapdh reverse, 5′-TTTGAGGGTGCAGCGAACTT-3′. fied [10, 11]. Monocarboxylate transporter 8 (MCT8; The relative mRNA expression levels were normal- Slc16a2) is highly specific for TH and has been shown ized against Gapdh expression. Real-time PCR analy- to be involved in the secretion of TH from thyrocytes sis was carried out at least in triplicate and repeated at [12, 13]. Cytosolic nicotinamide adenine dinucleo- least three times. tide phosphate (NADPH)-regulated TH binding pro- tein, also known as μ-crystallin (CRYM), has been Protein preparation and Western blotting identified as one of the TH-binding proteins involved Preparation of cell protein and Western blot analy- in intracellular TH transport [14], although its role in sis were performed as described [2, 6, 7]. Briefly, cells the thyrocyte is not clear. Therefore, we evaluated the were lysed in a buffer containing 50 mM HEPES, 150 effects of Tg on the expression levels of MCT8 and mM NaCl, 5 mM EDTA, 0.1% NP40 and 20% glyc- CRYM as specific TH transporters to elucidate the role erol, followed by addition of protease inhibitor cock- of accumulated follicular Tg in TH secretion. tail tablets (Complete Mini; Roche Diagnostics, Basel, Switzerland) at 4°C for 1 h. The mixture was centri- Materials and Methods fuged at 4°C for 20 min to recover cell protein. The protein concentration was determined using DC protein Cell culture and treatment assay reagents (BIO-RAD, Hercules, CA, USA) and a Rat thyroid FRTL-5 cells were grown in Coon’s VMax Kinetic Microplate Reader (Molecular Devices, modified Ham’s F-12 medium supplemented with 5% Sunnyvale, CA, USA) according to the manufacturer’s bovine serum (Invitrogen, Waltham, MA, USA) and a instructions. Western blotting was performed using 10 six-hormone mixture (1 mU/mL TSH, 10 μg/mL insu- μg of protein extracts separated on NuPage 4-12% Bis- lin, 10 ng/mL somatostatin, 0.36 ng/mL hydrocorti- Tris gels (Invitrogen) and transferred to PVDF mem- sone, 5 μg/mL transferrin, and 2 ng/mL glycyl-L-his- branes as described previously [2, 6, 7]. tidyl-L-lysine acetate) as described [2, 6, 15]. Culture The membranes were washed with PBS containing medium that did not contain TSH was also used in 0.1% Tween 20 (PBST), blocked with PBST contain- some experiments. Bovine Tg (Sigma-Aldrich, St. ing 5% nonfat milk for 1 h, and then incubated with Louis, MO, USA) was used at a final concentration of rabbit anti-NIS antibody (provided by Dr. N. Carrasco; 0.1-10 mg/mL [2, 3]. Bovine serum albumin (BSA) dilution 1:1000), rabbit anti-MCT8 antibody (LSBio, (Sigma-Aldrich) at the same concentrations as Tg was Seattle, WA, USA; 1:1000), rabbit anti-CRYM anti- used as a control. body (Proteintech, Chicago, IL, USA; 1:200), or goat anti-β-actin antibody (Santa Cruz Biotechnology, Total RNA isolation and real-time PCR Dallas, TX, USA; 1:2000) at 4°C for 12 h. After Total RNA was purified using RNeasy Plus Mini washing with PBST, membranes were incubated with Kit (Qiagen, Hilden, Germany), and cDNA was syn- either donkey anti-rabbit IgG-biotin conjugates (GE thesized using the High-Capacity cDNA Reverse Healthcare, Little Chalfont, UK 1:2000) or donkey Transcription Kits (Applied Biosystems, Foster City, anti-goat IgG-biotin conjugates (Millipore, Bellerica, CA, USA) as described previously [6]. Real-time PCR MA, USA; 1:2000) for 1 h. After washing, the mem- was performed using Fast SYBR Green Master Mix branes were incubated with streptavidin horseradish (Applied Biosystems) and StepOnePlus Real-Time peroxidase (GE Healthcare; 1:20000) for 1 h. Specific PCR System (Applied Biosystems) according to the bands were visualized using Immunostar LD reagent manufacturer’s instructions. The sequences of PCR (Wako, Osaka, Japan) and analyzed using a C- DiGit primers were as follows: blot scanner (LI-COR, Lincoln, NE) according to the Slc5a5 forward, 5′-CTACCGTGGGTGGTATGAAGG-3′; manufacturer’s instructions. Slc5a5 reverse, 5′-TGCCACCCACTATGAAAGTCC-3′; Slc16a2 forward, 5′-AACATGCGTGTATTTCGCCAGC-3′; Double immunofluorescence staining Slc16a2 reverse, 5′-GCAGGAATGAGAGGACCTGCAAG-3′; Paraffin-embedded rat thyroid sections were depa- Crym forward, 5′-AAGGAGGTGAGAATGTGGAACC-3′; raffinized in xylene and rehydrated through ethanol, Tg induces genes for hormone secretion 1009 followed by washing with deionized H2O. Antigen modified Slc16a2 expression (Fig. 1C and 1D), the retrieval was performed by heating the sections in effect of Tg was clearly stronger than that of BSA (Fig. 0.001 N NaOH at 121°C in an autoclave for 5 min as 1C, 48 h: p <0.001, 72 h: p < 0.001, Fig. 1D, 5 mg/mL: described [15-17]. The sections were blocked with p < 0.01, Fig. 1D, 10 mg/mL: p < 0.001). PBST containing 5% donkey serum (Cosmo Bio Co, We then examined the changes in the levels of NIS, Tokyo, Japan) for 1 h and incubated simultaneously
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